Biosynthetic proline/alanine random coil polypeptides and their uses

ABSTRACT

The present invention relates to a biosynthetic random coil polypeptide or a biosynthetic random coil polypeptide segment or biosynthetic conjugate, in which the biosynthetic random coil polypeptide, the biosynthetic random coil polypeptide segment, or the biosynthetic conjugate comprises an amino acid sequence consisting solely of proline and alanine amino acid residues, wherein the amino acid sequence consists of at least about 50 proline (Pro) and alanine (Ala) amino acid residues. The at least about 50 proline (Pro) and alanine (Ala) amino acid residues may be (a) constituent(s) of a heterologous polypeptide or an heterologous polypeptide construct. Also uses and methods of use of these biosynthetic random coil polypeptides or polypeptide segments or conjugates are described.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/697,569, which entered U.S. National Phase on Nov. 13, 2012 fromInternational Application No. PCT/EP2011/058307, filed May 20, 2011;which claims priority to Provisional U.S. Application 61/428,016, filedDec. 29, 2010; and European Patent Office Priority Application10163564.7, filed May 21, 2010. The foregoing applications areincorporated herein by reference in their entirety.

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety.

The present invention relates to a biosynthetic random coil polypeptideor a biosynthetic random coil polypeptide segment or a conjugate, saidbiosynthetic random coil polypeptide or a biosynthetic random coilpolypeptide segment or a conjugate comprising an amino acid sequenceconsisting solely of proline and alanine amino acid residues, whereinsaid amino acid sequence consists of at least about 50 proline (Pro) andalanine (Ala) amino acid residues. Said at least about 50 proline (Pro)and alanine (Ala) amino acid residues may be (a) constituent(s) of aheterologous polypeptide or an heterologous polypeptide construct. Alsouses and methods of use of these biosynthetic random coil polypeptides,said polypeptide segments or said conjugates are described. The usesmay, inter alia, comprise medical uses, diagnostic uses or uses in thefood industry as well as other industrial applications, like in thepaper industry, in oil recovery and the like. The present inventionrelates, also, to (a) specific use(s) of the herein providedbiosynthetic random coil polypeptide or biosynthetic random coilpolypeptide segment or conjugates, said biosynthetic random coilpolypeptide or biosynthetic random coil polypeptide segment orconjugates comprising an amino acid sequence consisting solely ofproline and alanine amino acid residues. The amino acid sequence of theherein provided biosynthetic random coil polypeptide or biosyntheticrandom coil polypeptide segment consists of at least about 50, of atleast about 100, of at least about 150, of at least about 200, of atleast about 250, of at least about 300, of at least about 350 or of atleast about 400 proline (Pro) and alanine (Ala) amino acid residues.Said at least about 50, at least about 100, at least about 150, at leastabout 200, at least about 250, at least about 300, at least about 350 orat least about 400 proline (Pro) and alanine (Ala) amino acid residuesare preferably (a) a constituent of a heterologous polypeptide or aheterologous polypeptide construct or are preferably (b) a constituentof a conjugate, like a drug conjugate, like a conjugate with a food orcosmetic ingredient or additive, like a conjugate with a biologicallyactive compound or like a conjugate with a spectroscopically activecompound. In particular, heterologous proteins are provided hereinwhereby these proteins comprise at least two domains, wherein a firstdomain of said at least two domains comprises an amino acid sequencehaving and/or mediating an activity, like a biological activity, and asecond domain of said at least two domains comprising the biosyntheticrandom coil proline/alanine polypeptide or proline/alanine polypeptidesegment of the present invention. The present invention relates inparticular to a drug conjugate comprising (i) a biosynthetic random coilpolypeptide or polypeptide segment comprising an amino acid sequenceconsisting solely of proline and alanine amino acid residues, whereinsaid amino acid sequence consists of at least 50 proline (Pro) andalanine (Ala) amino acid residues, and (ii) a drug selected from thegroup consisting of (a) a biologically active protein or a polypeptidethat comprises or that is an amino acid sequence that has or mediates abiological activity and (b) a small molecule drug. A further subject ofthe present invention is a drug conjugate comprising the biosyntheticrandom coil proline/alanine polypeptide or proline/alanine polypeptidesegment as provided herein and, additionally, (a) pharmaceutically ormedically useful molecule(s), like small molecules, peptides orbiomacromolecules (such as proteins, nucleic acids, carbohydrates, lipidvesicles) and the like, linked and/or coupled to said biosyntheticrandom coil proline/alanine polypeptide or proline/alanine polypeptidesegment. Furthermore, nucleic acid molecules encoding the biosyntheticrandom coil polypeptide or polypeptide segment and/or the biologicallyactive, heterologous proteins as well as vectors and cells comprisingsaid nucleic acid molecules are disclosed. Furthermore, methods for theproduction of the herein described inventive biosynthetic random coilpolypeptides or polypeptide segments and corresponding drug or foodconjugates, i.e.conjugates comprising the herein defined biosyntheticrandom coil polypeptides or polypeptide segments and a food ingredientor a food additive, are disclosed. Also disclosed are correspondingconjugates (comprising as one constituent the herein disclosedbiosynthetic random coil polypeptide or polypeptide segment) whichcomprise, inter alia, a cosmetic ingredient or additive or abiologically or spectroscopically active compound. In addition, thepresent invention provides compositions comprising the compounds of theinvention (i.e. the herein disclosed the random coil polypeptides orrandom coil polypeptide segments comprising an amino acid sequenceconsisting solely of proline and alanine amino acid residues and nucleicacid molecules encoding the same) as well as specific uses of saidrandom coil polypeptide or polypeptide segment, of the biologicallyactive proteins comprising said random coil polype random coilpolypeptides or random coil polypeptide segments ptides or random coilpolypeptide segments, the drug conjugates, the food conjugates or thenucleic acid molecules, vectors and cells of the invention. Also methodsof producing and/or obtaining the inventive biosynthetic random coilpolypeptides or polypeptide segments as well as of producing and/orobtaining the inventive biologically active, heterologous proteins,and/or polypeptide constructs or drug conjugates are provided. Inaddition, medical, pharmaceutical as well as diagnostic uses areprovided herein for the biosynthetic random coil polypeptide orpolypeptide segment comprising an amino acid sequence consisting solelyof proline and alanine amino acid residues (or for molecules andconjugates comprising the same) as defined herein. Such a medical orpharmaceutical use can comprise the use of said biosynthetic random coilpolypeptide or polypeptide segment as plasma expander and the like.However, the means and methods provided herein are not limited topharmaceutical, medical and biological uses but can also be employed inother industrial areas, like in the paper industry, in oil recovery,etc.

Rapid clearance from blood circulation by renal filtration is a typicalproperty of small molecules (including small proteins and peptides).However, by expanding the apparent molecular dimensions beyond the poresize of the kidney glomeruli plasma half-life of therapeutic proteinscan be extended to a medically useful range of several days. Onestrategy to achieve such an effect is chemical conjugation of thebiologic with the synthetic polymer poly-ethylene glycol (PEG). This hasled to several approved drugs, for example PEG-interferon alpha2a(Pegasys®), PEG-G-CSF (Neulasta®) and, recently, a PEGylatedalphaTNF-Fab fragment (Cimzia®). Nevertheless, the “PEGylation”technology has several drawbacks: clinical grade PEG derivatives areexpensive and their covalent coupling to a recombinant protein requiresadditional downstream processing and purification steps, thus loweringyield and raising the costs. Furthermore, PEG is not biodegradable,which can cause side effects such as vacuolation of kidney epitheliumupon continuous treatment; see, e.g., Gaberc-Porekar (2008) Curr OpinDrug Discov Devel 11:242-50; Knop (2010) Angew Chem Int Ed Engl49:6288-308 or Armstrong in: Veronese (Ed.), “PEGylated Protein Drugs:Basic Science and Clinical Applications”; Birkhäuser Verlag, Basel 2009.

In order to overcome some of the drawbacks of PEG technology, certainrecombinant polypeptide mimetics have been provided in the art, some ofwhich are based on naturally occurring amino acid sequences or syntheticamino acid stretches.

Most natural amino acid sequences do not behave like an ideal randomchain in physiological solution because they either tend to adopt afolded conformation (secondary structure) or, if unfolded, they usuallyare insoluble and form aggregates. In fact, most of the classicalexperiments to investigate the random chain behaviour of polypeptideswere conducted under denaturing conditions, i.e. in the presence ofchemical denaturants like urea or guanidinium chloride (see, e.g.,Cantor (1980) Biophysical Chemistry. W.H. Freeman and Company, NewYork). Hence, such technologies generally rest upon peculiar amino acidsequences that resist folding, aggregation as well as unspecificadsorption and, thus, provide stable random chains under physiologicalbuffer conditions and temperature even if genetically fused to a foldedtherapeutic protein domain. Under these circumstances, such recombinantPEG mimetics can confer a size increase much larger than one wouldnormally expect on the basis of their molecular mass alone, eventuallyretarding kidney filtration and effectively extending plasma half-lifeof the attached biologic by considerable factors.

A lot of these technologies have, however, further caveats anddisadvantages.

For example, naturally occurring repetitive amino acid sequences havebeen tested for their usefulness in medical sciences and inpharmaceutical approaches. One of these approaches relates to thetrans-sialidase of Trypanosoma cruzi. It contains a 680 amino acidresidue catalytic domain followed by a C-terminal repetitive domain,dubbed “shed acute phase antigen” (SAPA), which comprises a variablenumber of 12 mer amino acid repeats. Pharmacokinetic (PK) studies inmice of the trans-sialidase containing 13 hydrophilic and (atphysiological pH) negatively charged corresponding amino acid repeatshaving the natural sequence DSSAHSTPSTPA revealed a five-fold longerplasma half-life compared to the recombinant enzyme from which theC-terminal repetitive sequence had been deleted (Buscaglia (1999) Blood93:2025-32). A similar half-life extending effect was observed afterfusion of the same trans-sialidase, i.e. its 76 kDa catalytic domain,with 13 charged amino acid repeats of the sequence EPKSA that were foundin the Trypanosoma cruzi protein antigen 13. Both the repeats from SAPAand from the antigen 13 were able to prolong the plasma half-life of theheterologous protein gluthatione S-transferase (GST) from Schistosomajaponicum by a factor 7-8 after genetic fusion to both C-termini of thishomo-dimeric enzyme (see Buscaglia, loc. cit.). Yet, while thesenaturally occurring repetitive amino acid sequences from human pathogensin principle may appear attractive to optimize the pharmacokinetics oftherapeutic proteins they were found to be highly immunogenic (seeAffranchino (1989), Mol Biochem Parasitol 34:221-8 or Buscaglia (1998),J Infect Dis 1998; 177:431-6).

Another approach relates to the use of gelatin. Gelatin, hydrolyzed anddenatured animal collagen, contains long stretches of Gly-Xaa-Yaarepeats, wherein Xaa and Yaa mostly constitute proline and4-hydroxyproline, respectively. Succinylation of gelatin, primarily viathe ε-amino groups of naturally interspersed lysine side chains,increases the hydrophilicity of this biopolymer and lowers itsisoelectric point (pI). The intramolecular electrostatic repulsionbetween the negatively charged carboxylate groups of the modified sidechains supposedly spreads out the molecule into a more or less extendedconformation. The resulting expanded volume makes succinylated gelatin amacromolecule for use as plasma expander in humans and is, inter alia,marketed as Volplex® (Beacon Pharmaceuticals Ltd) or Gelofusine® (B.Braun Melsungen AG). Furthermore, a half-life extending effect wasachieved by genetic fusion of granulocyte-colony-stimulating factor(G-CSF) to an artificial gelatin-like polypeptide (Huang (2010) Eur JPharm Biopharm 74:435-41). To this end, all hydrophobic side chains in anatural gelatin were exchanged by hydrophilic residues, resulting in a116 amino acid gelatin-like protein (GLK) comprising the amino acids G,P, E, Q, N, S, and K in varying order. G-CSF was fused at its N-terminuswith 4 copies of this GLK sequence and secreted in Pichia pastoris.Pichia pastoris appeared as a favourable production organism for GLKfusion proteins; yet, if GLKs can also be produced in other organismsremains to be determined as it is known that recombinant gelatinfragments can be expressed with only low yield in E. coli, for example,as illustrated in Olsen (2003), Adv Drug Deliv Rev 55:1547-67.

Elastin is a component of the extracellular matrix in many tissues. Itis formed from the soluble precursor tropoelastin, which consists of ahydrophilic Lys/Ala-rich domain and a hydrophobic, elastomeric domainwith repetitive sequence. Enzymatic crosslinking of lysine side chainswithin the hydrophilic domain leads to insoluble elastin formation.Elastin-like polypeptides (ELPs) are artifically designed, repetitiveamino acid sequences derived from the hydrophobic domain oftropoelastin. The most common repeat sequence motif of ELPs isV-P-G-X-G, wherein “X” can be any amino acid except Pro (MacEwan (2010)Biopolymers 94:60-77; Kim (2010) Adv Drug Deliv Rev 62:1468-78).Suitable ELPs can be fused with therapeutic proteins and produced in E.coli. Consequently, the ability of ELPs to form gel-like depots afterinjection can significantly prolong the in vivo half-life of an attachedbiologic, albeit by a mechanism different from the other unstructuredpolypeptides. Yet, ELP attachment can hamper the bioactivity of thefusion partner as demonstrated for the interleukin-1 receptor antagonistin an IL-1-induced lymphocyte proliferation bioassay (Shamji (2007)Arthritis Rheum. 11:3650-3661). In addition, ELPs are subject todegradation by endogenous proteases such as collagenase. Also,aggregated proteins are generally more susceptible to immunogenicity.

Further approaches relate to the use of polyanionic polymers. Forexample, polyglutamate (PG) has been chemically coupled to poorlysoluble cytotoxic small molecule drugs for cancer treatment. Acorresponding product would be Opaxio™, a paclitaxel drug conjugatecurrently in clinical phase III studies. Half-life of a paclitaxel PGconjugate was prolonged by a factor 3 to 14 in comparison with theunmodified compound (Singer (2005) J Control Release 109:120-6). Furtherfusion proteins, for example G-CSF fused at its N-terminus with astretch of 175 consecutive Glu residues or IFN-alpha2 carrying at itsC-terminus a PG tail of 84 residues, were produced in a soluble state inthe cytoplasm of E. coli (see WO2002/077036). For efficient translation,the N-terminal fusion required a leader peptide, which was later removedby Tobacco Etch Virus (TEV) protease cleavage. Polyglutamate fusions ofG-CSF and INFα2 showed bioactivity in cell culture assays. However, todate no pharmacokinetic data of these PG fusions have been reported.Also, the highly negative charge of PG fusions is a general disadvantagewith respect to biomolecular interactions (e.g. binding of the targetreceptor or soluble factor) due to artificial electrostatic attractionor repulsion effects.

WO 2006/081249 describes a polypeptide sequence with about 2 to 500repeat units of 3 to 6 amino acids, wherein G, N or Q represent themajor constituents while minor constituents can be A, S, T, D or E. Thisamino acid composition allows integration of the glycosylation sequonAsn-Xaa-Ser/Thr (where Xaa is any amino acid except Pro) for N-linkedglycosylation of the Asn side chain in eukaryotic expression systems.The increased macromolecular size of a resulting fusion protein,including posttranslational modification with bulky solvatedcarbohydrate structures, can extend the pharmacokinetics of thegenetically conjugated protein. Such oligosaccharide attachments(“glycoengineering”) in general can both reduce susceptibility toproteolysis and increase the hydrodynamic volume (Sinclair (2005) JPharm Sci 94:1626-35). A disadvantage is the intrinsic molecularheterogeneity of the glycosylated biomacromolecule, which causesadditional effort during biotechnological production and qualitycontrol.

WO 2010/091122 (and WO 2007/103515) and Schellenberger (2009) NatBiotechnol 27:1186-90 disclose unstructured non-repetitive amino acidpolymers encompassing and comprising the residues P, E, S, T, A and G.This set of amino acids, which shows a composition not unlike the PSTADrepeat described further above, was systematically screened forsequences to yield a solvated polypeptide with large molecular size,suitable for biopharmaceutical development, by avoiding hydrophobic sidechains—in particular F, I, L, M, V and W—that can give rise toaggregation and may cause an HLA/MHC-II mediated immune response. Also,potentially crosslinking Cys residues, the cationic amino acids K, R andH, which could interact with negatively charged cell membranes, and theamide side chains of N and Q, which are potentially prone to hydrolysis,were excluded (see Schellenberger (2009) loc. cit.). Synthetic genelibraries encoding non-repetitive sequences comprising the PESTAG set ofresidues, which were fused to the green fluorescent protein (GFP), werescreened with respect to soluble expression levels in E. coli, and aresulting subset was further investigated for genetic stability, proteinsolubility, thermostability, aggregation tendency, and contaminantprofile. Eventually, an 864 amino acid sequence containing 216 Serresidues (25.0 mole %), 72 Ala residues (8.3 mole %) and 144 amino acids(16.7 mole %) of each Pro, Thr, Glu, and Gly was further tested forfusion to the GLP-1 receptor agonist Exendin-4 (E-XTEN) and a few otherbiologics. The fusion proteins—typically carrying a cellulose bindingdomain, which was later cleaved off—were produced in a soluble state inthe cytoplasm of E. coli and isolated. Investigation of of E-XTEN bycircular dichroism (CD) spectroscopy revealed lack of secondarystructure while during size exclusion chromatography (SEC) the fusionprotein showed substantially less retention than expected for a 84 kDaprotein, thus demonstrating an increased hydrodynamic volume(Schellenberg (2009) loc. cit.). The disordered structure of the PESTAGpolypeptide and the associated increase in hydrodynamic radius may befavoured by the electrostatic repulsion between amino acids that carry ahigh net negative charge which are distributed across the XTEN sequence(see WO 2010/091122). However, a further study Geething (2010) PLoS One2010; 5:e10175 demonstrated that XTEN decreases potency of itstherapeutic fusion partner. In a cell culture assay, a glucagon XTENfusion showed merely 15% bioactivity of the non-modified peptide. Aneven stronger loss in receptor affinity (17-fold increased EC₅₀) wasdescribed for an XTEN fusion of human growth hormone (hGH); see WO2010/144502.

Also glycine, as the smallest and structurally simplest amino acid, hasbeen considered as the conformationally most flexible amino acid basedon theoretical grounds; see, e.g. Schulz G E, Schirmer R H. Principlesof Protein Structure. Springer, N.Y. 1979. Furthermore, computersimulations have indicated that Gly polymers lack secondary structureand are likely to form a random coil in solution; see Shental-Bechor(2005) Biophys J 88:2391-402. From a chemical perspective, polyglycineis a linear unbranched polyamide that shows certain resemblance to thepolyether PEG in so far as both are essentially one-dimensionalmacromolecules with many rotational degrees of freedom along the chain,which are made of repeated short hydrocarbon units that are regularlyinterrupted by hydrogen-bonding and highly solvated polar groups.Consequently, polyglycine should constitute the simplest geneticallyencodable PEG mimetic with prospects for extending the plasma half-lifeof therapeutic proteins. This concept was employed in form of“homo-amino-acid polymer (HAP)” or as glycine rich sequence (GRS),respectively; see, Schlapschy (2007) Protein Eng Des Sel 20:273-84; WO2007/103515. However, it has long been known that chemically synthesizedpure polymers of Gly show poor solubility in water; see, inter alia, inBamford C H et al. Synthetic Polypeptides—Preparation, Structure, andProperties. Academic Press, New York 1956. Hence, different attemptswere made to increase hydrophilicity, either by introducinghydrogen-bonding serine alcohol side chains (WO 2007/103515 as well asSchlapschy (2007) loc. cit.) or, in addition, negatively chargedglutamate residues (WO 2007/103515). It is of note that peptide spacerswith the composition (Gly₄Ser)_(n) have already been described in theart in order to link domains in fusion proteins in a flexible manner. Asignificantly increased hydrodynamic volume was detected for thesefusion proteins in analytical SEC. In the case of the 200 residue HAPversion the apparent size increase was 120% compared with the unfusedFab fragment, while the true mass was only bigger by 29%, hencerevealing the effect of an enhanced hydrodynamic volume due to thesolvated random coil structure of the polyglycine tag. Furthermore, CDdifference spectra were characteristic for disordered secondarystructure for the HAP moiety. Finally, terminal plasma half-life of theFab fragment carrying the 200 residue HAP in mice was prolonged byapproximately a factor 3. Though moderate, this effect could beappropriate for certain (specialized) diagnostic applications, such asin vivo imaging; see Schlapschy (2007); loc. cit. Unfortunately, theproduction of fusion proteins with longer (Gly₄Ser)_(n) repeat sequencesappeared less feasible due to an increasing tendency to form aggregates,thus posing a natural limitation to the use of—more or less pure—glycinepolymers as PEG mimetics.

WO 2008/155134 discloses that sequences with an appropriate mixture ofPro, Ala, and Ser (i.e. PAS) residues lead to mutual cancellation oftheir distinct secondary structure preferences and, thus, result in astably disordered polypeptide. However, WO 2008/155134 also documentsthat fusion proteins with a domain composed only of serine and alanine(SA) residues, i.e. a domain comprising only two types of amino acids,do not form a random coil, but a β-sheet structure instead.

The chemical synthesis of polypeptides is well known and has beendescribed in the art. Izuka discloses the chemical synthesis ofpolypeptides containing proline (see Izuka (1993), Bull. Chem. Soc. Jpn66, 1269-1272). These copolypeptides contain random sequences of prolineand either glycine, L-alanine, L-α-aminobutyric acid (Abu), L-norvaline(Nva) or L-leucine, respectively, and are synthesized by chemicalcopolymerization. Izuka discloses that such copolypeptides mostly have adefined collagen-like conformation. Further, it is described in thispublication that copolypeptides of proline and alanine (or proline andL-α-aminobutyric acid) are partially soluble in water, while othercopolypeptides were completely insoluble. It is speculated in Izuka thatproline/alanine copolypeptides may have a partial disorderedconformation. Izuka emphasizes that chemically synthesized polypeptideswith a random proline/alanine sequence occur predominantly in acollagen-like conformation, i.e. in a structured conformation.

Thus, the technical problem underlying the present invention is theprovision of large polypeptides with true random coil conformation. Thetechnical problem is solved by provision of the embodimentscharacterized in the claims and as provided herein.

Accordingly, the invention relates to the provision and use of abiosynthetic random coil polypeptide or polypeptide segment comprisingan amino acid sequence consisting of at least about 50, in particular ofat least about 100, in particular of at least about 150, in particularof at least about 200, in particular of at least about 250, inparticular of at least about 300, in particular of at least about 350,in particular of at least about 400 proline and alanine amino acidresidues. The invention therefore relates to the provision ofbiosynthetic random coil polypeptides or polypeptide segments comprisingan amino acid sequence of at least 50 amino acid residues, said aminoacid sequence consisting solely of proline and alanine amino acidresidues and comprising at least one proline and at least one alanine.The invention also provides for a drug conjugate comprising (i) abiosynthetic random coil polypeptide or polypeptide segment comprisingan amino acid sequence consisting solely of proline and alanine aminoacid residues, wherein said amino acid sequence consists of at least 50proline (Pro) and alanine (Ala) amino acid residues, and (ii) a drugselected from the group consisting of (a) a biologically active proteinor a polypeptide that comprises or that is an amino acid sequence thathas or mediates a biological activity and (b) a small molecule drug. Thepolypeptides with true random coil conformation and polypeptide segmentswith true random coil conformation as provided herein are also useful inthe context of cosmetic uses as well as uses in food industry and theproduction of beverages. The large polypeptides provided herein whichshow true random coil confirmation consist soley and merely of proline(P, Pro) and alanine (A, Ala) residues and comprise more than at least50 amino acids, in particular of at least about 100, in particular of atleast about 150, in particular of at least about 200, in particular ofat least about 250, in particular of at least about 300, in particularof at least about 350, in particular of at least about 400 proline andalanine amino acid residues. Both amino acids, P and A, need to bepresent in the herein provided large polypeptides with true random coilconformation and polypeptide segments with true random coilconformation. Also provided herein are nucleic acid molecules thatencode for the herein disclosed biosynthetic random coil polypeptides orpolypeptide segments as well as for drug or food conjugates thatcomprise said biosynthetic random coil polypeptides or polypeptidesegments and a (covalently linked) protein of interest, like abiologically active protein.

The biosynthetic random coil polypeptide or biosynthetic random coilpolypeptide segment as described herein and to be used in drug or foodconjugates as provided herein and comprising an amino acid sequenceconsisting of at least about 50, of at least about 100, of at leastabout 150, of at least about 200, of at least about 250, of at leastabout 300, of at least about 350, of at least about 400 proline (P) andalanine (A) amino acid residues is, inter alia, to be used in aheterologous context, i.e. in a biologically active heterologousprotein, protein construct and/or in a drug conjugate comprising saidbiosynthetic random coil polypeptide or polypeptide segment andpharmaceutically or medically useful molecules, like small molecules,peptides or biomacromolecules such as proteins, nucleic acids,carbohydrates, lipid vesicles and the like. As illustrated in theappended examples, the inventors could successfully provide for drugconjugates which consist of the true random coil polypeptides as definedherein and biologically active proteins or protein stretches as well asdrug conjugates that consist of small molecules or small molecule drugsthat comprise and/or are linked to the herein described random coilpolypeptides, consisting solely of proline and alanine amino acidresidues (i.e. of both amino acids P and A).

Accordingly, the present invention provides, inter alia, for abiologically active, heterologous protein comprising at least twodomains wherein (a) a first domain of said at least two domainscomprises an amino acid sequence having and/or mediating said biologicalactivity; and (b) a second domain of said at least two domains comprisesthe biosynthetic random coil polypeptide or polypeptide segmentconsisting of an amino acid sequence consisting of at least about 50, ofat least about 100, of at least about 150, of at least about 200, of atleast about 250, of at least about 300, of at least about 350, of atleast about 400 proline and alanine amino acid residues. In accordancewith this invention, said “first domain” and said “second domain” arenot comprised in either a natural (i.e. occurring in nature) protein ora hypothetical protein as deduced from naturally occurring codingnucleic acid sequences, like open reading frames etc.

Furthermore, this invention provides for a drug conjugate consisting ofthe biosynthetic random coil polypeptide or polypeptide segmentcomprising an amino acid sequence consisting of at least about 50, of atleast about 100, of at least about 150, of at least about 200, of atleast about 250, of at least about 300, of at least about 350, of atleast about 400 proline and alanine amino acid residues and (a)pharmaceutically, therapeutically and/or medically useful molecule(s),like (a) small molecule(s), (a) peptide(s) or (a) biomacromolecule(s)such as protein(s), a nucleic acid(s), (a) carbohydrate(s), (a) lipidvesicle(s) and the like, that is/are conjugated to said biosyntheticrandom coil polypeptide or polypeptide segment. Again, it is of notethat the term “biologically active” in context of herein disclosedconjugates is not limited to pure biological molecules but also comprisemedically active, therapeutically active, pharmacuticlly activemolecules and the like. It is evident for the skilled artisan that themeans and methods provided herein are not limited to pharmaceutical andmedical uses, but can be employed in a wide variety of technologies,including, but not limited to cosmetic, food, beverage and nutritiontechnologies, oil industry, paper industry and the like.

In contrast to chemically synthesized copolypeptides (like in Izuka,loc. cit.), the random coil polypeptides provided herein arebiosynthetically produced. The term “biosynthetic” as used herein refersto the synthesis by means of biotechnological methods (in contrast tochemical synthesis). Such biotechnological methods are well known in theart and also described herein further below. The biosynthesis of therandom coil polypeptides of the present invention allows the productionof polypeptides with a defined sequence of proline and alanine residues,a defined length and/or a defined ratio of proline and alanine residues.Further, the polypeptides provided in accordance with the presentinvention are substantially pure, i.e. the produced polypeptides areessentially uniform and share the above characteristics (i.e. definedsequence, defined length and/or defined amino acid ratio). The randomcoil polypeptides consisting of at least about 50, in particular of atleast about 100, in particular of at least about 150, in particular ofat least about 200, in particular of at least about 250, in particularof at least about 300, in particular of at least about 350, inparticular of at least about 400 proline and alanine amino acid residuesare, in accordance with this invention for example comprised inbiologically active, heterologous polypeptides/polypeptide constructsand/or in drug or food conjugates as well as in other conjugates usefulin further industrial areas, like, but not limited to paper industry,oil industry and the like.

Overall, the above features of the polypeptides of the present inventionpermit the formation of a stable random coil of the polypeptides andthese random coil polypeptides have surprising and advantageousproperties. For example, the polypeptides of the present invention arecompletely soluble in aqueous solution and have an increasedhydrodynamic volume. Unexpectedly, the random coil polypeptides asdefined herein are also capable of conferring an increased in vivo/invitro stability. This is particularly important for medicalapplications, for example, for biologically active proteins or drugconjugates comprising the random coil polypeptide of this invention.However, the numerous advantageous properties of the random coilpolypeptides of the present invention not only permit their use in themedical field but also in other fields, like in cosmetics/cosmetictreatments or in the fields of nutrition and food technology, like inthe dairy industry or in meat processing. Examples of conjugates usefulin food industry and the like are conjugates that comprise the hereindisclosed random coil polypeptide or polypeptide segment comprising anamino acid sequence consisting solely of proline and alanine amino acidresidues and compounds that are useful in these technologies, like, e.g.polyoxypropylene or polyoxyethylene polymers, which are non-ionicsurfactants used as emulsifiers. Also envisaged herein is the use of thebiosynthetic random coil polypeptide as defined herein in biochemicalmethods and in technical processes, such as paper production, oilrecovery and the like. The surprising and advantageous characteristicsof the biosynthetic random coil polypeptides consisting merely ofproline and alanine residues as provided herein (and as also of theherein disclosed conjugates and constructs, like drug or foodconjugates/constructs, comprising said biosynthetic, true random coilpolypeptides) are described below in greater detail. Furthermore,illustrative uses and means and methods employing these inventivebiosynthetic random coil polypeptides are provided below. Also means andmethods for the production of such biosynthetic random coil polypeptidesas well as biologically active, heterologous polypeptides or polypeptideconstructs and of the herein dislosed conjugates and constructs, likedrug constructs, comprising said random coil polypeptides are providedherein.

In context of this invention, it has been surprisingly found thatproline-alanine polymers/polypeptides with a uniform composition formstable random coil conformation. This is also demonstrated in theappended examples, where random coil structure of biosyntheticproline/alanine (co)-polymers/polypeptides is confirmed by circulardichroism (CD) spectroscopy. Obtaining and employing such biosynthetic,truly random coil polypeptides/polymers was surprising since theestablished Chou-Fasman method (Chou and Fasman (1974), Biochemistry 13,223-245) predicts a 100% α-helical secondary structure ofpolymers/polypeptides (or segments thereof) composed of proline andalanine, as shown in FIG. 7. Yet, herein it has been surprisingly foundand experimentally shown that proline-alanine polymers/polypeptides witha uniform composition form a stable random coil conformation. This isalso demonstrated in the appended examples, where random coil structureof proline/alanine (co)-polymers/polypeptides is confirmed byexperimental techniques like circular dichroism (CD) spectroscopy andsize exclusion chromatography (SEC).

In contrast to the polypeptides/polymers of the present invention, thechemically synthesized polypeptides described, for example, in Izuka(1993), loc. cit. have an arbitrary/undefined and stochastic sequenceand a diverse length. Thus, the chemically synthesized polypeptidescomprise a mixture of completely different peptides with variousproline/alanine ratios, lengths, and so on. As mentioned in Izuka, thechemically synthesized polypeptides of such a mixture do not (or onlypartially) form a random coil and, accordingly, do not have any of theadvantageous properties of the biosynthetic polypeptides provided anddescribed herein below. Accordingly, the present invention comprises andrelates to compositions comprising the inventive biosynthetic randomcoil polypeptides/polymers as disclosed herein, whereby saidbiosynthetic random coil polypeptides/polymers are defined, inter alia,by their sequence comprising solely proline and alanine residues. In oneparticular embodiment, the present invention relates to conjugates, likedrug or food conjugates comprising, as one constituent, these randomcoil polypeptides/polymers disclosed herein. These inventivebiosynthetic random coil polypeptides/polymers comprised in saidcompositions are, in one embodiment, of uniform length.

As mentioned above, the biosynthetic random coil polypeptides (or randomcoil polypeptide segments) of this invention consisting solely ofproline and alanine residues unexpectedly form a stable random coilconformation. The term “random coil” as used herein relates generally toany conformation of a polymeric molecule, including amino acidpolymers/amino acid sequences/polypeptides, in which the individualmonomeric elements that form said polymeric structure are essentiallyrandomly oriented towards the adjacent monomeric elements while stillbeing chemically bound to said adjacent monomeric elements. Inparticular, a polypeptide, amino acid sequence or amino acid polymeradopting/having/forming “random coil conformation” substantially lacks adefined secondary and tertiary structure. In context of the polypeptidesof the present invention, the monomeric elements forming the polymericstructure (i.e. the polypeptide/amino acid sequence) are either singleamino acids such as proline and alanine per se or peptide stretches suchas the “amino acid repeats”/“amino acid cassettes”/“cassetterepeats”/“building blocks”/“modules” (or fragments thereof) which aredescribed and defined further below.

The nature of polypeptide random coils and their methods of experimentalidentification are known to the person skilled in the art and have beendescribed in the scientific literature (Cantor (1980) BiophysicalChemistry, 2nd ed., W.H. Freeman and Company, New York; Creighton (1993)Proteins-Structures and Molecular Properties, 2nd ed., W.H. Freeman andCompany, New York; Smith (1996) Fold Des 1:R95-R106). The term “segment”as used herein refers to a part of the herein defined biosyntheticrandom coil polypeptide, whereby such a part may be an internal part ofthe biosynthetic random coil polypeptide described herein. Such a“segment” may be, for example, a biosynthetic random coil polypeptide asdefined herein where one (or more) amino acid(s) has/have been deleted,e.g. from the start and/or from the end of the polypeptide of theinvention. Furthermore, such a “segment” may be used as or may form partof a larger protein or polypeptide, for example, of a fusion proteinwith a biologically active protein. Such a “fusion protein” would alsobe an example of a heterologous, biologically activepolypeptide/protein/polypeptide construct of the present invention. Theterm “heterologous” as used herein is defined herein below.

The random coil polypeptide (or random coil segment thereof), asprovided in the present invention and to be employed in context of thisinvention, adopts/forms random coil conformation, for example, inaqueous solution or at physiological conditions. The term “physiologicalconditions” is known in the art and relates to those conditions in whichproteins usually adopt their native, folded conformation. Morespecifically, the term “physiological conditions” relates to thebiophysical parameters as they are typically valid for higher forms oflife and, particularly, in mammals, most preferably human beings. Theterm “physiological conditions” may relate to the biochemical andbiophysical parameters as they are normally found in the body (inparticular in body fluids) of mammals and in particular in humans. Said“physiological conditions” may relate to the corresponding parametersfound in the healthy body as well as the parameters found under diseaseconditions or in human patients. For example, a sick mammal or humanpatient may have a higher, yet “physiological” temperature conditionwhen said mammal or said human suffers from fever. With respect to“physiological conditions” at which proteins adopt their nativeconformation/state, the most important parameters are temperature (37°C. for the human body), pH (7.35-7.45 for human blood), osmolarity(280-300 mmol/kg H₂O), and, if necessary, protein content (66-85 g/lserum). Yet, the person skilled in the art is aware that atphysiological conditions these parameters may vary, e.g. thetemperature, pH, osmolarity, and protein content may be different ingiven body or tissue fluids such as blood, liquor cerebrospinalis,peritoneal fluid and lymph (Klinke (2005) Physiologie, 5th ed., GeorgThieme Verlag, Stuttgart). For example, in the liquor cerebrospinalisthe osmolarity may be around 290 mmol/kg H₂O and the proteinconcentration may be between 0.15 g/l to 0.45 g/l while in the lymph thepH may be around 7.4 and the protein content may be between 3 g/l and 5g/l. When determining whether a polypeptide (or segment thereof)/aminoacid sequence forms/adopts random coil conformation under experimentalconditions using the methods as described herein below, the biophysicalparameters such as temperature, pH, osmolarity and protein content maybe different to the physiological conditions normally found in vivo.Temperatures between 1° C. and 42° C. or preferably 4° C. to 25° C. maybe considered useful to test and/or verify the biophysical propertiesand biological activity of a protein under physiological conditions invitro.

Several buffers, in particular in experimental settings (for example inthe determination of protein structures, in particular in CDmeasurements and other methods that allow the person skilled in the artto determine the structural properties of a protein/amino acid stretch)or in buffers, solvents and/or excipients for pharmaceuticalcompositions, are considered to represent “physiologicalsolutions”/“physiological conditions” in vitro. Examples of such buffersare, e.g. phosphate-buffered saline (PBS: 115 mM NaCl, 4 mM KH₂PO₄, 16mM Na₂HPO₄ pH 7.4), Tris buffers, acetate buffers, citrate buffers orsimilar buffers such as those used in the appended examples. Generally,the pH of a buffer representing “physiological solution conditions”should lie in a range from 6.5 to 8.5, preferably in a range from 7.0 to8.0, most preferably in a range from 7.2 to 7.7 and the osmolarityshould lie in a range from 10 to 1000 mmol/kg H₂O, more preferably in arange from 50 to 500 mmol/kg H₂O and most preferably in a range from 200to 350 mmol/kg H₂O. Optionally, the protein content of a bufferrepresenting physiological solution conditions may lie in a range from 0to 100 g/l, neglecting the protein with biological activity itself,whereby typical stabilizing proteins may be used, for example human orbovine serum albumin.

It has been found herein that the polypeptides (or segments thereof) notonly form random coil conformation under physiological conditions but,more generally, in aqueous solution. The term “aqueous solution” is wellknown in the art. An “aqueous solution” may be a solution with a water(H₂O) content of at least about 20%, of at least about 30%, of at leastabout 40%, of at least about 50%, of at least about 60%, of at leastabout 70%, of at least about 80% or of at least about 90% H₂O(weight/weight). Accordingly, the polypeptide (or segment thereof) ofthe present invention may form random coil conformation in aqueoussolution, possibly containing other miscible solvents, or in aqueousdispersions with a wider range of temperatures, pH values, osmolaritiesor protein content. This is particularly relevant for applications ofthe random coil polypeptide (or segment thereof) outside medical therapyor in vivo diagnostics, for example in cosmetics, nutrition or foodtechnology.

Accordingly, it is also envisaged in the context of this invention thatthe random coil conformation of the proline/alanine biosyntheticpolypeptide (or segment thereof) of the present invention is maintainedin and/or is used in context of pharmaceutical compositions, like liquidpharmaceuticals/biologicals or lyophilized pharmaceutical compositions.This is particularly important in context of the herein providedbiologically active, heterologous proteins or the drug conjugatescomprising, inter alia, the inventive random coil polypeptide (orpolypeptide segment). Preferably, “physiological conditions” are to beused in corresponding buffer systems, solvents and/or excipients. Yet,for example in lyophilized or dried compositions (like, e.g.pharmaceutical compositions/biologicals), it is envisaged that therandom coil conformation of the herein provided random coil polypeptide(or polypeptide segment) is transiently not present and/or cannot bedetected. However, said random coil polypeptide (or polypeptide segment)will adopt/form again its random coil after reconstitution incorresponding buffers/solutions/excipients/solvents or afteradministration to the body. Methods for determining whether apolypeptide (or segment thereof) forms/adopts random coil conformationare known in the art (Cantor (1980) loc. cit.; Creighton (1993) loc.cit.; Smith (1996) loc. cit.). Such methods include circular dichroism(CD) spectroscopy as exemplified herein below. CD spectroscopyrepresents a light absorption spectroscopy method in which thedifference in absorbance of right- and left-circularly polarized lightby a substance is measured. The secondary structure of a protein can bedetermined by CD spectroscopy using far-ultraviolet spectra with awavelength between approximately 190 and 250 nm. At these wavelengths,the different secondary structures commonly found in polypeptides can beanalyzed, since α-helix, parallel and anti-parallel β-sheet, and randomcoil conformations each give rise to a characteristic shape andmagnitude of the CD spectrum. Accordingly, by using CD spectrometry theskilled artisan is readily capable of determining whether polypeptide(or segment thereof) forms/adopts random coil conformation in aqueoussolution or at physiological conditions. Other established biophysicalmethods include nuclear magnetic resonance (NMR) spectroscopy,absorption spectrometry, infrared and Raman spectroscopy, measurement ofthe hydrodynamic volume via size exclusion chromatography, analyticalultracentrifugation or dynamic/static light scattering as well asmeasurements of the frictional coefficient or intrinsic viscosity(Cantor (1980) loc. cit.; Creighton (1993) loc. cit.; Smith (1996) loc.cit.).

In addition to the experimental methods above, theoretical methods forthe prediction of secondary structures in proteins have been described.One example of such a theoretical method is the Chou-Fasman method (Chouand Fasman, loc. cit.) which is based on an analysis of the relativefrequencies of each amino acid in α-helices, β-sheets, and turns basedon known protein structures solved, for example, with X-raycrystallography. However, theoretical prediction of protein secondarystructure is known to be unreliable. As exemplified herein below, aminoacid sequences expected to adopt an α-helical secondary structureaccording to the Chou-Fasman method were experimentally found to form arandom coil. Accordingly, theoretical methods such as the Chou-Fasmanalgorithm may only have limited predictive value whether a givenpolypeptide adopts random coil conformation, as also illustrated in theappended examples and figures. Nonetheless, the above describedtheoretical prediction is often the first approach in the evaluation ofa putative secondary structure of a given polypeptide/amino acidsequence. A theoretical prediction of a random coil structure also oftenindicates that it might be worthwhile verifying by the aboveexperimental means whether a given polypeptide/amino acid sequence hasindeed a random coil conformation.

Homo-polymers of most amino acids, in particular the hydrophobic aminoacids, are usually insoluble in aqueous solution (Bamford (1956)Synthetic Polypeptides—Preparation, Structure, and Properties, 2nd ed.,Academic Press, New York). Homo-polymers of several hydrophilic aminoacids are known to form secondary structures, for example α-helix in thecase of Ala (Shental-Bechor (2005) Biophys J 88:2391-2402) and β-sheetin the case of Ser (Quadrifoglio (1968) J Am Chem Soc 90:2760-2765)while poly-proline, the stiffest homooligopeptide (Schimmel (1967) ProcNatl Acad Sci USA 58:52-59), forms a type II trans helix in aqueoussolution (Cowan (1955) Nature 176:501-503).

Using the theoretical principles of polymer biophysics the random coildiameter of a chain of 200 amino acid residues would amount in the caseof poly-glycine, for example, to ca. 75 Å—calculated as the average rootmean square end-to-end distance of

=l·√{square root over (n·C_(∞))}, with n=200 rotatable bonds of lengthl=3.8 Å for each Cα-Cα distance and the ‘characteristic ratio’ C_(∞)≈2.0for poly(Gly) (Brant (1967) J Mol Biol 23:47-65; Creighton, (1993) loc.cit.). This relation shows that the person skilled in the art wouldexpect that the hydrodynamic volume of a random chain amino acid polymercan be either extended by (a) using a longer chain length l or by (b)using amino acids that exhibit a larger characteristic ratio, C₂₈. C₂₈is a measure for the inherent stiffness of the molecular random chainand has a general value of 9 for most amino acids (Brant (1967) loc.cit.). Only Gly, which lacks a side chain, and also the imino acid Proexhibit significantly smaller values. Hence, Gly and Pro (underdenaturing conditions) are expected to contribute to reducing thedimensions of random coil proteins (Miller (1968) Biochemistry7:3925-3935) Amino acid sequences comprising proline residues,accordingly, are expected to have a relatively compact hydrodynamicvolume. In contrast to this teaching, however, it is shown herein thatthe hydrodynamic volume of the amino acid polymers/polypeptides of theinvention that comprise a mixture of proline and alanine residues have adramatically increased hydrodynamic volume as determined by analyticalgel permeation/size exclusion chromatography when compared to theexpected hydrodynamic volume. In fact, it is surprising thatpolypeptides comprising mixtures of these two amino acids (proline andalanine), of which each alone tends to form a homooligopeptide withdefined secondary structure, adopt random coil conformation underphysiological conditions. Such inventive proline/alanine polypeptideshave a larger hydrodynamic radius than homo-polymers comprising the samenumber of Gly residues, for example, and they confer better solubilityto the biologically active proteins or constructs, i.e. biologicallyactive heterologous proteins or drug conjugates, according to theinvention.

As mentioned above, the biosynthetic random coil proline/alaninepolypeptides of the present invention differ from chemically synthesizedpolypeptides in that they can adopt a defined, uniform length by easymeans and methods. Whereas the prior art provides mixtures/compositionsof polypeptides with enormous variations in terms of the length of thepeptides, the present invention can provide mixtures/compositions ofbiosynthetic random coil polypeptides with a defined length. Preferably,essentially all polypeptides of the invention comprised in such amixture/composition have the same defined length, and, hence, share thesame biochemical characteristics. Such a uniform composition is moreadvantageous in the various medical, cosmetic, nutritional applications,wherein the biosynthetic random coil polypeptides can be employed.Furthermore, in particular in a medical or pharmaceutical context, theherein defined biosynthetic random coil polypeptide or polypeptidesegment comprising an amino acid sequence consisting of at least about50, in particular of at least about 100, in particular of at least about150, in particular of at least about 200, in particular of at leastabout 250, in particular of at least about 300, in particular of atleast about 350, in particular of at least about 400 proline and alanineamino acid residues can also be used in the prevention, ameliorationand/or treatment of disorders linked and/or affiliated with an impairedblood plasma situation, for example after injuries, burns, surgery andthe like. One medical use of said biosynthetic random coil polypeptidesor polypeptide segments is, accordingly, the use as plasma expander.However, it is of note that in accordance with this invention also theherein described drug conjugates and heterologous polypeptides orheterologous polypeptide constructs may be employed in context of themedical or pharmaceutical intervention of a disorder related to animpaired blood plasma amount or blood plasma content or of a disorderrelated to an impaired blood volume.

Accordingly, the present invention relates in one embodiment to abiosynthetic random polypeptide (or segment thereof) which comprises anamino acid sequence consisting solely of at least about 50 proline andalanine amino acid residues, of at least about 100 proline and alanineamino acid residues, of at least about 150 proline and alanine aminoacid residues or of at least about 200 proline and alanine residues, inparticular when comprised in a heterologousprotein/polypeptide/polypeptide construct or in a drug conjugate. Thepresent invention also relates to biosynthetic random coil polypeptideswhich comprise an amino acid sequence consisting solely of at leastabout 200 proline and alanine amino acid residues, even more preferablyof at least about 300 proline and alanine amino acid residues,particularly preferably of at least about 400 proline and alanine aminoacid residues, more particularly preferably of at least about 500proline and alanine amino acid residues and most preferably of at leastabout 600 proline and alanine amino acid residues. The amino acidsequence forming random coil conformation may consist of maximally about3000 proline and alanine amino acid residues, of maximally about 2000proline and alanine amino acid residues, of maximally about 1500 prolineand alanine amino acid residues, of maximally about 1200 proline andalanine amino acid residues, of maximally about 800 proline and alanineamino acid residues. Accordingly, the proline/alanine amino acidsequence stretch may consist of about 50, of about 100, of about 150, ofabout 200, of about 250, of about 300, of about 350, of about 400, ofabout 500, of about 600, of about 700, of about 800, of about 900 toabout 3000 proline and alanine amino acid residues. In certainembodiments, the inventive biosynthetic amino acid sequence comprisesabout 200 to about 3000 proline and alanine residues, about 200 to about2500 proline and alanine residues, about 200 to about 2000 proline andalanine residues, about 200 to about 1500 proline and alanine residues,about 200 to about 1000 proline and alanine residues, about 300 to about3000 proline and alanine residues, about 300 to about 2500 proline andalanine residues, about 300 to about 2000 proline and alanine residues,about 300 to about 1500 proline and alanine residues, about 300 to about1000 proline and alanine residues, about 400 to about 3000 proline andalanine residues, about 400 to about 2500 proline and alanine residues,about 400 to about 2000 proline and alanine residues, about 400 to about1500 proline and alanine residues, about 400 to about 1000 proline andalanine residues, about 500 to about 3000 proline and alanine residues,about 500 to about 2500 proline and alanine residues, about 500 to about2000 proline and alanine residues, about 500 to about 1500 proline andalanine residues, about 500 to about 1000 proline and alanine residues,about 600 to about 3000 proline and alanine residues, about 600 to about2500 proline and alanine residues, about 600 to about 2000 proline andalanine residues, about 600 to about 1500 proline and alanine residues,about 600 to about 1000 proline and alanine residues, about 700 to about3000 proline and alanine residues, about 700 to about 2500 proline andalanine residues, about 700 to about 2000 proline and alanine residues,about 700 to about 1500 proline and alanine residues, about 700 to about1000 proline and alanine residues, about 800 to about 3000 proline andalanine residues, about 800 to about 2500 proline and alanine residues,about 800 to about 2000 proline and alanine residues, about 800 to about1500 proline and alanine residues, about 800 to about 1000 proline andalanine residues. As is evident from the content of this invention, alsolarger biosynthetic amino acid sequences (consisting essentially ofproline and alanine) are within the scope of this invention and canreadily be employed in the herein defined biologically active proteinsor protein constructs which comprise as one domain of at least twodomains an amino acid sequence having and/or mediating said biologicalactivity and as another domain of at least two domains the biosyntheticrandom coil polypeptide or polypeptide segment consisting of at leastabout 50 proline and alanine amino acid residues, of at least about 100proline and alanine amino acid residues, of at least about 150 prolineand alanine amino acid residues, of at least about 200, of at leastabout 250, of at least about 300, of at least about 350, of at leastabout 400 proline and alanine amino acid residues. Such a biosyntheticrandom coil polypeptide or polypeptide segment corresponds to thebiosynthetic random coil part of a heterologous protein/proteinconstruct. These biosynthetic proline/alanine stretches consist ofmaximally about 3000 proline and alanine amino acid residues. Theseamino acid sequences (proline/alanine stretches) comprise proline andalanine as main or unique residues as explained further below.

It is envisaged that it is the herein defined biosynthetic amino acidsequence consisting solely of proline (P) and alanine (A) amino acidresidues, which forms/adopts/has a random coil conformation. In thesimplest case, the biosynthetic polypeptide or polypeptide segmentconsists of the amino acid sequence having a random coil conformation asdefined herein. However, the biosynthetic polypeptide (or segmentthereof) may, in addition to the herein described amino acid sequenceforming/adopting/having a random coil conformation, comprise furtheramino acid sequences/amino acid residues which do not contribute to theformation of the random coil conformation or which are not capable offorming/adopting/having a random coil conformation on their own. Withoutdeferring from the gist of the invention, also such biosyntheticpolypeptides (or segments thereof) are biosynthetic “random coil”polypeptides or polypeptide segments. The further amino acidsequences/amino acid residues may, for example, be useful as linkers.Inter alia, dimers, trimers, i.e. in general multimers of thebiosynthetic random coil polypeptide are also envisaged in context ofthe present invention and such multimers may be linked by amino acidsequences/residues which do not form random coil conformation. Anexample of a protein which may comprise such a random coil polypeptideis the herein provided biologically active protein, which may, inaddition to the random coil polypeptide consisting of proline andalanine amino acid residues as defined herein further comprise anotherpolypeptide having/mediating biological activity. Again, such aconstruct may be a heterologous, biologically active protein orpolypeptide construct as described herein.

The term “at least about 50/100/150/200/300/400/500/600/700/800/etcamino acid residues” is not limited to the concise number of amino acidresidues but also comprises amino acid stretches that comprise eitheradditional about 1-20%, like 10% to 20% residues or about 1-20%, likeabout 10% to 20% less residues. For example “at least about 100 aminoacid residues” may also comprise about 80 to 100 and about 100 to 120amino acid residues without deferring from the gist of the presentinvention. For example “at least about 200 amino acid residues” may alsocomprise about 160 to 200 and about 200 to 240 amino acid residueswithout deferring from the gist of this invention. The definition andexplanations given herein above, apply, mutatis mutandis, also to theterm “maximally about 3000/2000/1500/1200/800 amino acid residues” etc.Accordingly, the term “about” is not limited or restricted to theconcise number of amino acid residues in context of longer amino acidsequences (e.g. amino acid sequences comprising or consisting ofmaximally 3000 amino acid residues). Therefore, the term “maximallyabout 3000/2000/1500/1200/800 amino acid residues” but may also compriseamino acid stretches that comprise additional 10% to 20% or 10% to 20%less residues without deferring from this invention.

Furthermore, the biosynthetic random coil polypeptides (or segmentsthereof) are characterised by a defined content or ratio of amino acidresidues, in particular of the main constituents proline and alanine. Asmentioned above, the present invention relates to a biosynthetic randomcoil polypeptide or polypeptide segment comprising an amino acidsequence consisting solely of proline and alanine amino acid residues,wherein said amino acid sequence consists of at least about 50, of atleast about 100, of at least about 150, of at least about 200 of atleast about 250, of at least about 300, of at least about 350, of atleast about 400 proline (Pro) and alanine (Ala) amino acid residues inparticular when comprised in a heterologous biological activeprotein/protein construct/polypeptide or drug conjugate. The term“solely” as used herein means that preferably at least about 90% or atleast about 95% of the amino acids are proline and alanine, wherebyproline and alanine constitute the majority but may not be the onlyamino acid residues, i.e. these inventive amino acid sequences are notnecessarily 100% proline and alanine amino acid stretches. Hence, thebiosynthetic polypeptides/amino acid sequences of the present inventionmay also comprise other amino acids than proline and alanine as minorconstituents as long as the amino acid sequence forms/adopts/has randomcoil conformation. Such a random coil conformation can be easilydetermined by herein provided means and methods. Accordingly, also incontext of the term “solely”, a minor amount (less than about 10% orless than about 5%) of other amino acid residues may be comprised. Said“other”, minor amino acid residues are defined herein below.

Accordingly, the present invention relates in one embodiment to abiosynthetic random coil polypeptide (or segment thereof) whereby theamino acid sequence consists mainly of proline and alanine, and whereinthe proline residues constitute more than about 10% and less than 75% ofthe amino acid sequence. The alanine residues comprise the remaining atleast 25% to 90% of said amino acid sequence (or the random coilpolypeptide or polypeptide segment if it consists of the amino acidsequence).

Preferably, the amino acid sequence comprises more than about 10%,preferably more than about 12%, even more preferably more than about14%, particularly preferably more than about 18%, more particularlypreferably more than about 20%, even more particularly preferably morethan about 22%, 23% or 24% and most preferably more than about 25%proline residues. The amino acid sequence preferably comprises less thanabout 75%, more preferably less than 70%, 65%, 60%, 55% or 50% prolineresidues, wherein the lower values are preferred. Even more preferably,the amino acid sequence comprises less than about 48%, 46%, 44%, 42%proline residues. Particular preferred are amino acid sequencescomprising less than about 41%, 40%, 39% 38%, 37% or 36% prolineresidues, whereby lower values are preferred. Most preferably, the aminoacid sequence comprise less than about 35% proline residues; see alsothe herein below provided PA constructs.

Vice versa, the amino acid sequence preferably comprises less than about90%, more preferably less than 88%, 86%, 84%, 82% or 80% alanineresidues, wherein the lower values are preferred. Even more preferably,the amino acid sequence comprises less than about 79%, 78%, 77%, 76%alanine residues, whereby lower values are preferred. Most preferably,the amino acid sequence comprises less than about 75% alanine residues.

Also preferred herein is an amino acid sequence comprising more thanabout 25%, preferably more than about 30%, even more preferably morethan about 35%, particularly preferably more than about 40%, moreparticularly preferably more than about 45% or 50%, even moreparticularly preferably more than about 52%, 54%, 56%, 58% or 59%alanine residues, wherein the higher values are preferred. Even morepreferably, the amino acid sequence comprises more than about 60%, 61%,62%, 63% or 64% alanine residues and most preferably more than about 65%alanine residues.

Accordingly, the random coil polypeptide (or segment thereof) maycomprise an amino acid sequence consisting of about 25% prolineresidues, and about 75% alanine residues. Alternatively, the random coilpolypeptide (or segment thereof) may comprise an amino acid sequenceconsisting of about 35% proline residues and about 65% alanine residues.The term “about X %” as used herein above is not limited to the concisenumber of the percentage, but also comprises values of additional 10% to20% or 10% to 20% less residues. For example the term 10% may alsorelate to 11% or 12% and to 9% and 8%, respectively.

However, as mentioned above and further detailed herein below saidrandom coil polypeptide (or polypeptide segment), and, in particular theamino acid sequence, may also comprise additional amino acids differingfrom proline and alanine as minor constituents. As already discussedherein above, said minor constituent(s), i.e. (an)other amino acid(s)than proline or alanine, may comprise less than about 10%, less thanabout 9%, less than about 8%, less than about 7%, less than about 6%,less than about 5%, less than about 4%, less than about 4%, less thanabout 3% or less than about 2% of the biosynthetic random coilpolypeptide/polymer of this invention.

The skilled person is aware that an amino acid sequence/polypeptide (orsegment thereof) may also form random coil conformation when otherresidues than proline and alanine are comprised as a minor constituentin said amino acid sequence/polypeptide (polypeptide segment). The term“minor constituent” as used herein means that maximally 5% or maximally10% amino acid residues are different from proline or alanine in theinventive biosynthetic random coil polypepitdes/polymers of thisinvention. This means that maximally 10 of 100 amino acids may bedifferent from proline and alanine, preferably maximally 8%, i.e.maximally 8 of 100 amino acids may be different from proline andalanine, more preferably maximally 6%, i.e. maximally 6 of 100 aminoacids may be different from proline and alanine, even more preferablymaximally 5%, i.e. maximally 5 of 100 amino acids may be different fromproline and alanine, particularly preferably maximally 4%, i.e.maximally 4 of 100 amino acids may be different from proline andalanine, more particularly preferably maximally 3%, i.e. maximally 3 of100 amino acids may be different from proline and alanine, even moreparticularly preferably maximally 2%, i.e. maximally 2 of 100 aminoacids may be different from proline and alanine and most preferablymaximally 1%, i.e. maximally 1 of 100 of the amino acids that arecomprised in the random coil polypeptide (or segment thereof) may bedifferent from proline and alanine. Said amino acids different fromproline and alanine, may be selected from the group consisting of Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp,Tyr, and Val, including posttranslationally modified amino acids ornon-natural amino acids (see, e.g., Budisa (2004) Angew Chem Int Ed Engl43:6426-6463 or Young (2010) J Biol Chem 285:11039-11044). In case thatthe “minor constituent” (i.e. an amino acid other than proline andalanine) of the biosynthetic random coil polypeptide/construct/polymer(or a fragment thereof) comprises as “other amino acid”/“different aminoacid” (a) Ser(s), said Ser amino acid/Ser amino acids constitutepreferably less than 50%, more preferably less than 40%, less than 30%,less than 20% or less than 10% of these (minor) amino acid residues. Ina most preferred embodiment, the biosynthetic random coilpolypeptide/construct/polymer as described herein or the random coilpolypeptide part of a (e.g.) fusion protein as described herein does notcomprise (a) serine residue(s). It is, generally, preferred herein thatthese “minor” amino acids (other than proline and alanine) are notpresent in the herein provided biosynthetic random coilpolypeptide/construct/polymer as described herein or the random coilpolypeptide part of a (e.g.) fusion protein. In accordance with theinvention, a biosynthetic random coil polypeptide (or segmentthereof)/the amino acid sequence may, in particular, consist exclusivelyof proline and alanine amino acid residues (i.e. no other amino acidresidues are present in the random coil polypeptide or in the amino acidsequence).

Whereas the above relates to the overall length and proline/alaninecontent of the amino acid sequence comprised in the random coilpolypeptide (or segment thereof), the following relates in greaterdetail to the specific, exemplary amino acid sequences (or fragmentsthereof).

In one embodiment, the amino acid sequences/polypeptides adopting randomcoil conformation (the random coil polypeptide or segment thereof asdefined herein), for example, in aqueous solution or under physiologicalconditions may comprise a plurality of “amino acid repeats”/“amino acidcassettes”/“cassette repeats”, wherein said “amino acid repeats”/“aminoacid cassettes”/“cassette repeats”/“building block”/“modules” (theseterms are used herein interchangeably) mainly or exclusively consist ofproline (Pro, P) and alanine (Ala, A) amino acid residues (depictedherein as “PA”, or as “AP”), wherein no more than 6 consecutive aminoacid residues are identical. An illustrative “building block” is, e.g.“AP” and this has also been provided in the appended illustrativeexamples as functional biosynthetic random coil domain of the presentinvention. This illustrative example is the sequence “P1A1” as alsoprovided in form of APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 51). i.e. a “polyPA” “amino acid repeat”/“amino acid cassette”/“cassette repeat”. In apreferred embodiment, the amino acid sequence/polypeptide comprising theabove defined “amino acid repeats”/“amino acid cassettes”/“cassetterepeats” and the like comprises no more than 5 identical consecutiveamino acid residues. Other alternative embodiments are provided hereinbelow in context of exemplified, individual building blocks.

Within a random coil polypeptide (or segment thereof) according to thisinvention the amino acid repeats may be identical or non-identical.Non-limiting examples of “amino acid repeats”, “building blocks”,“modules”, “repeats”, “amino acid cassettes” etc. consisting of prolineand alanine residues are provided herein below; see, e.g. SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 andSEQ ID NO. 51 (The enclosed sequence listing also comprises illustrativenucleic acid sequences which encode such “repeats”/“modules”, etc. Theappended sequences in said sequence listing as filed herewith constitutepart of this specification and description). Also the use of (identicaland/or non-identical) fragments of these sequences is envisasged herein,whereby a “fragment” comprises at least 2 amino acids and comprises atleast one proline and/or alanine, preferably at least one proline andone alanine. “Fragments” of these sequences to be employed in accordancewith this invention for the generation of the random coil polypeptide(or segment thereof) may consist of at least 3, preferably of at least4, more preferably of at least 5, even more preferably of at least 6,still more preferably of at least 8, particularly preferably of at least10, more particularly preferably of at least 12, even more particularlypreferably of at least 14, still more particularly preferably of atleast 16, and most preferably of at least 18 consecutive amino acids ofthe amino acid sequence selected from the group consisting of said SEQID NOs: 1, 2, 3, 4, 5, 6 and 51 (here it is of note that SEQ ID No. 51consists of an illustrative “AP” or “PA” repeat).

Based on the teaching given herein, the person skilled in the art isreadily in a position to generate further amino acidsequences/polypeptides that form random coil conformation for exampleunder aqueous or under physiological conditions and are constituted ofmainly proline and alanine as defined herein. Further examples of randomcoil conformation forming amino acid sequences/polypeptides to be usedas building blocks or modules of the herein defined random coilpolypeptide (or segment thereof) may, inter alia, comprise combinationsand/or fragments or circularly permuted versions of the specific“building blocks”, “polymer cassettes”, or “polymer repeats” shownabove. Accordingly, the exemplified modules/sequence units/polymerrepeats/polymer cassettes of the random coil polypeptide/amino acidsequence may also provide for individual fragments which may be newlycombined to form further modules/sequence units/polymer repeats/polymercassettes in accordance with this invention.

The terms “module(s)”, “sequence unit(s)”, “polymer repeat(s)”, “polymercassette(s)” and “building block(s)” are used as synonyms herein andrelate to individual amino acid stretches which may be used to form theherein defined random coil polypeptide (or segment thereof)/amino acidsequence.

An amino acid repeat (used as “building block” etc. of a biosyntheticrandom coil polypeptide of the present invention) may consist of atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues,wherein each repeat comprises (an) proline and alanine residue(s).However, as illustrated in appended SEQ ID No. 51, said “building block”can also merely consist of the 2 herein provided amino acid residues Pand A, namely in form of “PA” or “AP”. In one embodiment, the amino acidrepeat according to the present invention does not comprise more than 50amino acid residues. However, it is evident for the skilled artisan thatsuch a “repeat” may comprise even more than 50 amino acid residues, forexample in cases wherein said inventive biosynthetic random coilpolypeptide/polymer comprises more than about, e.g., 100 amino acids,more than about 150 amino acids, more than about 200 amino acids, etc.Accordingly, the maximal amount of amino acid residues comprised in sucha “repeat” is conditioned by the over-all length of the biosyntheticpolypeptide (or segment thereof)/polymer as provided herein.

Yet, it is of note that the biosynthetic random coil polypeptides/aminoacid sequences comprising the above repeats etc. should preferably havethe overall length and/or proline/alanine content as defined andexplained herein above, i.e. consist of about 50, of about 100, of about150, of about 200, of about 250, of about 300, of about 350, of about400 to about 3000 amino acids and/or comprise more than about 10% andless than about 75% proline residues. All the definitions given hereinabove in this context also apply here, mutatis mutandis.

As discussed in detail herein and as provided herein above, the presentinvention provides for (a) biologically active, heterologous protein(s)or (a) protein construct(s) that is/are particularly useful in apharmaceutical, medical and/or medicinal setting. These biologicallyactive, heterologous proteins/protein constructs comprise as at leastone domain of said at least two domains the random coil polypeptide orpolypeptide segment comprising an amino acid sequence consisting solelyof proline and alanine residues, wherein said amino acid sequenceconsist of about 50, of about 100, of about 150, of about 200, of about250, of about 300, of about 350, of about 400 to about 3000 proline(Pro) and alanine (Ala) residues.

In context of the biologically active, heterologous proteins,polypeptides or protein constructs as disclosed herein, the term“heterologous” relates to at least two domains within said proteins,polypeptides or protein constructs wherein a first of said at least twodomains confers, has and/or mediates a defined biological activity andwherein a second of said at least two domains comprises the biosyntheticrandom coil polypeptide consisting solely of proline and alanine aminoacid residues and whereby said at least two domains are not foundoperationally linked to each other in nature or are not encoded by asingle coding nucleic acid sequence (like an open reading frame)existing in nature. The biosynthetic random coil polypeptide/polypeptidesegment consisting solely of proline and alanine amino acid residues asprovided herein and as employed in the biologically active, heterologousproteins/protein constructs of this invention are preferably not further(chemically) modified, for example they are preferably neitherglycosylated nor hydroxylated.

It is of note that certain naturally occurring proteins or hypotheticalproteins as deduced from sequenced nucleic acid stretches found innature are described as comprising a relatively high (i.e. aboveaverage) content of proline and alanine. For example, a homologoushypothetical protein has been described for Leishmania major strainFriedlin (Ivens (2005) Science 309, 436-442.). The disclosed readingframe comprising 1514 codon triplets includes a stretch of 412 tripletscomposed of 240 Ala, 132 Pro, 34 Lys and 4 Val codons. The Lys residues,which are positively charged under physiological buffer conditions, arealmost evenly distributed among this sequence, suggesting a solubilizingeffect. However, as is evident from the disclosure herein, such ahomologous hypothetical protein as deduced from a naturally occurringnucleic acid molecule or open reading frame, comprising a high prolineand alanine content above average is not part of this invention. Theinvention is based on the fact that a rather large random coilpolypeptide or polypeptide segment that does not occur in nature in anisolated manner and that comprises an amino acid sequence consistingsolely of proline and alanine residues, wherein said amino acid sequenceconsist of about 50, of about 100, of about 150, of about 200, of about250, of about 300, of about 350, of about 400 to about 3000 proline(Pro) and alanine (Ala) residues is provided that is particularly usefulin medical/pharmaceutical context. The herein described isolatedbiosynthetic random coil polypeptides or polypeptide segments that donot occur in nature in an isolated manner are also comprised in theherein disclosed (a) biologically active, heterologous protein(s) or (a)protein construct(s) that is/are particularly useful in apharmaceutical, medical and/or medicinal setting. These biologicallyactive, heterologous proteins/protein constructs comprise as at leastone domain of said at least two domains the random coil polypeptide orpolypeptide segment comprising an amino acid sequence consisting solelyof proline and alanine residues, wherein said amino acid sequenceconsists of about 50, of about 100, of about 150, of about 200, of about250, of about 300, of about 350, of about 400 to about 3000 proline(Pro) and alanine (Ala) residues.

Also, arabinogalactan proteins (AGPs), Pro-rich proteins, and extensinsbelong to a large group of glycoproteins, known as hydroxyproline(Hyp)-rich glycoproteins (HRGPs), which are expressed throughout theplant kingdom. One such AGP motif comprising an Ala-Pro repeat (AP)51was expressed as a synthetic glycomodule peptide with N-terminal signalsequence and C-terminal green fluorescent protein in transgenicArabidopsis thaliana and investigated as a substrate for prolylhydroxylases and subsequent O-glycosylation of the hydroxyprolineresidues (Estevez (2006) Plant Physiol. 142, 458-470). Again, thedisclosed hydroxylated and/or glycosylated Pro side chains, which canform hydrogen bonds to water molecules, appear to have a solubilizingeffect.

It is of note that the herein described “biologically active proteins orprotein constructs comprising as (at least) one domain a biosyntheticrandom coil polypeptide or peptide segment comprising an amino acidsequence consisting solely of proline and alanine residues” relate toproteins or protein constructs that do not normally occur in nature and,thus, are “heterologous”. Furthermore, and in contrast to proline-richsequences described in the plant kingdom, the biosynthetic random coilpolypeptides/polypeptide segments described herein are preferably notchemically modified, i.e. they are preferably not glycosylated orhydroxylated.

A particular advantage of the biosynthetic random coil polypeptides orpolypeptide segments of this invention is their intrinsicallyhydrophilic but uncharged character. Accordingly, as “minor” amino acids(other than proline and alanine) in the herein described biosyntheticrandom coil polypeptide or polypeptide stretch such amino acids arepreferred that do not have hydrophobic side chains, like Val, Ile, Leu,Met, Phe, Tyr or Trp, and/or that do not have charged side chains, likeLys, Arg, Asp or Glu. In accordance with this invention, it is envisagedthat (in cases where such individual amino acids are neverthelesscomprised in the inventive biosynthetic random coilpolypeptide/polypeptide segment) the overall content of each individualamino acid having a hydrophobic side chain, like Val, Ile, Leu, Met,Phe, Tyr or Trp, and/or having a charged side chain, like Lys, Arg, Asp,or Glu, within the herein defined biosynthetic random coil polypeptide(or segment thereof) does not exceed 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

The biosynthetic random coil polypeptide/amino acid sequences of thepresent invention may comprise concatamers of individual blockscomprising combined proline/alanine stretches of the sequence(Pro)_(x)-(Ala)_(y), whereby x can have an integer value from 1 topreferably 15, more preferably 1 to 10, even more preferably 1 to 5, andy can have an integer value from 1 to preferably 15, more preferably 1to 10, even more preferably 1 to5, and x and y can vary betweensubsequent blocks. Said x and y can also be an integer of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

The amino acid sequences/polypeptides forming random coil conformationin aqueous solution or under physiological conditions may have theformula (I):[Pro_(x)Ala_(y)]_(n)wherein x is independently selected from integer 1 to 5. Furthermore,for each n, y is independently selected from integer 1 to 5. n, finally,is any integer provided that random coil polypeptide (or segmentthereof)/amino acid sequence consists preferably of at least about 50,of at least about 100, of at least about 150, of at least about 200, ofat least about 250, of at least about 300, of at least about 350, of atleast about 400 amino acid residues and up to about 3000 amino acidresidues. Also in this context it is of note that the polypeptides/aminoacid sequences comprising the above concatemers or having the aboveformula (I) should preferably have the overall length and/orproline/alanine content as defined and explained herein above, i.e.consist of about 50, of about 100, of about 150, of about 200, of about250, of about 300, of about 350, of about 400 to about 3000 amino acidsand/or comprise more than about 10% and less than about 75% prolineresidues. Again, all the definitions given herein above in this contextalso apply here, mutatis mutandis.

The present invention also relates to random coil polypeptides((a)polypeptide segment(s))/amino acid sequences comprising an aminoacid stretch selected from the group consisting of AAPAAPAPAAPAAPAPAAPA(SEQ ID NO: 1); AAPAAAPAPAAPAAPAPAAP (SEQ ID NO: 2);AAAPAAAPAAAPAAAPAAAP (SEQ ID NO: 3 being an example for [Pro₁Ala₃]₅);AAPAAPAAPAAPAAPAAPAAPAAP (SEQ ID NO: 4); APAAAPAPAAAPAPAAAPAPAAAP (SEQID NO: 5); AAAPAAPAAPPAAAAPAAPAAPPA (SEQ ID NO: 6) andAPAPAPAPAPAPAPAPAPAP (SEQ ID NO: 51 being an example for [Pro₁Ala₁]₁₀)or circular permuted versions or (a) multimers(s) of these sequences asa whole or parts of these sequences. Accordingly, the random coilpolypeptide ((a) polypeptide segment(s) thereof)/amino acid sequence maycomprise the amino acid stretch AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 1),AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 1); AAPAAAPAPAAPAAPAPAAP (SEQ ID NO:2); AAAPAAAPAAAPAAAPAAAP (SEQ ID NO: 3); AAPAAPAAPAAPAAPAAPAAPAAP (SEQID NO: 4); APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO: 5);AAAPAAPAAPPAAAAPAAPAAPPA (SEQ ID NO: 6) and APAPAPAPAPAPAPAPAPAP (SEQ IDNO: 51), as well as combinations of these motifs or combinations offragments and parts of this motifs as long as the resulting biosyntheticrandom coil polypeptide consists solely of proline and alanine aminoacid residues, wherein said amino acid sequence consists of at least 50proline (Pro) and alanine (Ala) amino acid residues.

Also circular permuted versions of the above amino acid sequences may beused in accordance with the present invention. Exemplary circularpermuted versions of e.g. AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 1) can beeasily generated, for example by removing the first alanine and addinganother alanine at the end of the above sequence. Such a cicularpermuted version of SEQ ID NO: 1 would then be APAAPAPAAPAAPAPAAPAA (SEQID NO: 7). Further, non-limiting examples of cicular permuted versionsof SEQ ID NO. 1 are:

(SEQ ID NO: 8) PAAPAPAAPAAPAPAAPAAA, (SEQ ID NO: 9)AAPAPAAPAAPAPAAPAAAP, (SEQ ID NO: 10) APAPAAPAAPAPAAPAAAPA,(SEQ ID NO: 11) PAPAAPAAPAPAAPAAAPAA, (SEQ ID NO: 12)APAAPAAPAPAAPAAAPAAP, (SEQ ID NO: 13) PAAPAAPAPAAPAAAPAAPA,(SEQ ID NO: 14) AAPAAPAPAAPAAAPAAPAP, (SEQ ID NO: 15)APAAPAPAAPAAAPAAPAPA, (SEQ ID NO: 16) PAAPAPAAPAAAPAAPAPAA,and the like. Based on the teaching of the present invention, a skilledperson is easily in the position to generate corresponding circularpermuted versions of the amino acid stretches as shown in SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO. 51(said SEQ ID No. 51 being entirely based on “AP” repeats and a circularpermutated version could be based entirely on “PA” or “AP”repeats/building blocks).

Such circular permuted versions may also be considered as examples of afurther “module”/“building block” etc. of the herein providedpolypeptides/amino acid sequences which can be used accordingly herein.

It is evident for the person skilled in the art that also “modules” and(shorter) fragments or circularly permuted versions of the hereinprovided amino acid stretches may be used as “modules”, “repeats” and/orbuilding blocks for the herein defined random coil polypeptide (orsegment thereof)/amino acid sequence.

In accordance with the above, the random coil polypeptide/amino acidsequence forming random coil conformation may comprise a multimer of anyof the above amino acid stretches (or circular permuted versions orfragments thereof), preferably those shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO.51. It is to note that these sequences are by no means limiting incontext of this invention.

Again, the polypeptides/amino acid sequences comprising the above aminoacid stretches (or fragments thereof), circular mutated versions (orfragments thereof) should preferably have the overall length and/orproline/alanine content as defined and explained herein above, i.e.consist of about 50, of about 100, of about 150, of about 200, of about250, of about 300, of about 350, of about 400 to about 3000 amino acidsand/or comprise more than about 10% and less than about 75% prolineresidues. All the definitions given herein above in this context alsoapply here, mutatis mutandis. Also the term “fragment” has been definedabove.

As mentioned above, in context of this invention it was surprisinglyfound that the biosynthetic random coil polypeptides (or polypeptidesegment)/polymers as provided herein are characterized by a relativelylarge hydrodynamic volume. This hydrodynamic volume, also calledapparent size, can easily be determined by analytical gel filtration(also known as size exclusion chromatography, SEC). Preferably, therandom coil polypeptide (or segment thereof) has an apparent size of atleast 10 kDa, preferably of at least 25 kDa, more preferably of at least50 kDa, even more preferably of at least 100 kDa, particularlypreferably of at least 200 kDa and most preferably of at least 400 kDa.The person skilled in the art is readily capable of determining thehydrodynamic volume of specific proteins. Such methods may includedynamic/static light scattering, analytical ultracentrifugation oranalytical gel filtration as exemplified herein below. Analytical gelfiltration represents a known method in the art for measuring thehydrodynamic volume of macromolecules. Alternatively, the hydrodynamicvolume of a globular polypeptide can be estimated by its molecularweight (Creighton (1993) loc. cit.). As described herein thehydrodynamic volume of the polypeptides of the invention consistingpreferably of at least about 50, of at least about 100, of at leastabout 150, of at least about 200, of at least about 250, of at leastabout 300, of at least about 350, of at least about 400 to about 3000proline and alanine amino acid residues and having random coilconformation show unexpectedly high values in relation to thehydrodynamic volume that would be estimated for a corresponding folded,globular protein based on the molecular weight. The following relates tobiologically active, heterologous proteins or protein constructscomprising, inter alia, the biosynthetic random coil polypeptide (orsegment thereof) as described and defined herein above which represent aprefered embodiment of the present invention. Without being bound bytheory, it was surprisingly found in context of the present inventionthat the biosynthetic random coil polypeptide stretches as providedherein and consisting solely of proline and alanine can, even providefor a higher hydrodynamic volume than a corresponding biosyntheticrandom coil stretch having the same total number of amino acid residuesbut consisting solely of proline, alanine and serine (as provided in WO2008/155134).

Common human plasma proteins such as serum albumin (HSA) andimmunoglobulins (Igs), including humanized antibodies, show longhalf-lifes, typically of 2 to 3 weeks, which is attributable to theirspecific interaction with the neonatal Fc receptor (FcRn), leading toendosomal recycling (Ghetie (2002) Immunol Res, 25:97-113). In contrast,most other proteins of pharmaceutical interest, in particularrecombinant antibody fragments, hormones, interferons, etc. suffer fromrapid (blood) clearance. This is particularly true for proteins whosesize is below the threshold value for kidney filtration of about 70 kDa(Caliceti (2003) Adv Drug Deliv Rev 55:1261-1277). In these cases theplasma half-life of an unmodified pharmaceutical protein may beconsiderably less than one hour, thus rendering it essentially uselessfor most therapeutic applications. In order to achieve sustainedpharmacological action and also improved patient compliance—withrequired dosing intervals extending to days or even weeks—severalstrategies were previously established for purposes of biopharmaceuticaldrug development.

First, the recycling mechanism of natural plasma proteins has beenemployed by producing fusion proteins with the Fc portion of Igs, forexample Enbrel®, a hybrid between the extracellular domain of TNFαreceptor and human IgG1 (Goldenberg (1999) Clin Ther 21:75-87) or withserum albumin, for example Albuferon® (albinterferon alfa-2b, ZALBIN™,JOULFERON®), a corresponding fusion of IFNalpha with HSA (Osborn (2002)J Pharmacol Exp Ther 303:540-548). Albumin with its high plasmaconcentration of 600 μM has also been utilized in an indirect manner,serving as carrier vehicle for biopharmaceuticals that are equipped withan albumin-binding function, for example via fusion with a bacterialalbumin-binding domain (ABD) from Streptococcal protein G (Makrides(1996) J Pharmacol Exp Ther 277:534-542) or with a peptide selectedagainst HSA from a phage display library (Dennis (2002) J Biol Chem,277:35035-35043; Nguyen (2006) Protein Eng Des Sel 19:291-297).

Second, a fundamentally different methodology for prolonging the plasmahalf-life of biopharmaceuticals is the conjugation with highly solvatedand physiologically inert chemical polymers, thus effectively enlargingthe hydrodynamic radius of the therapeutic protein beyond the glomerularpore size of approximately 3-5 nm (Caliceti (2003) loc. cit.). Covalentcoupling under biochemically mild conditions with activated derivativesof polyethylene glycol (PEG), either randomly via Lys side chains (Clark(1996) J Biol Chem 271:21969-21977) or by means of specificallyintroduced Cys residues (Rosendahl (2005) BioProcess International:52-60) has been moderately successful and is currently being applied inseveral approved drugs. Corresponding advantages have been achievedespecially in conjunction with small proteins possessing specificpharmacological activity, for example Pegasys®, a chemically PEGylatedrecombinant IFNa-2a (Harris (2003) Nat Rev Drug Discov, 2:214-221; Walsh(2003) Nat Biotechnol 21:865-870).

However, the chemical coupling of a biologically active protein withsynthetic polymers has disadvantages with respect to biopharmaceuticaldevelopment and production. Suitable PEG derivatives are expensive,especially as high purity is needed, and their conjugation with arecombinant protein requires additional in vitro processing andpurification steps, which lower the yield and raise the manufacturingcosts. In fact, PEG is often contaminated with aldehydes and peroxides(Ray (1985) Anal Biochem 146:307-312) and it is intrinsically prone tochemical degradation upon storage in the presence of oxygen. Also, thepharmaceutical function of a therapeutic protein may be hampered ifamino acid side chains in the vicinity of its biochemical active sitebecome modified by the PEGylation process. Furthermore, chemicalcoupling with synthetic polymers usually results in a heterogeneousmixture of molecules which may show a substantial variance of in vivoactivity.

Third, the use of glycosylation analogs of biologically active proteinsin which new N-linked glycosylation consensus sequences are introducedhas been proposed to prolong plasma half-life; see WO 02/02597; Perlman(2003) J Clin Endocrinol Metab 88:2327-2335; or Elliott (2003) NatBiotechnol 21:414-420). The described glycoengineered proteins, however,displayed an altered in vivo activity, which indicates that the newcarbohydrate side chains influence the biological activity of theengineered protein. Moreover, the additional carbohydrate side chainsare likely to increase the antigenicity of the resulting biologicalactive molecules, which raises substantial safety concerns. Furthermore,fusion proteins comprising the Trypanosoma cruzi derived artificialrepetitive sequence PSTAD have been reported to induce a prolongedplasma half-life of trans-sialidase (Alvarez (2004) JBC 279:3375-3381).Yet, such Trypanosoma cruzi derived repeats have been reported to inducea humoral immune response (Alvarez (2004) loc. cit.). Accordingly,alternative strategies to prolong the action of biologically activeproteins are desired.

The biosynthetic amino acid sequences/polypeptides as disclosed hereinand consisting solely of proline and alanine according to the inventionwere surprisingly found to adopt random coil conformation in particularunder physiological conditions. Therefore, they are advantageousmolecules to provide for the herein below defined “second domain” of thebiologically active protein(s)/polypeptide(s), i.e. comprising apolypeptide stretch that forms under physiological conditions a randomcoil conformation and thereby mediates an increased in vivo and/or invitro stability to biologically active (“functional”) protein(s) orpolypeptide(s), in particular, an increased plasma half-life. Thehydrodynamic volume of a functional protein that is fused to said randomcoil domain is dramatically increased as can be estimated by usingstandard methods mentioned herein. Since the random coil domain isthought not to interfere with the biological activity of the firstdomain of the biologically active protein, the biological activitymediated by the functional protein of interest to which it is fused isessentially preserved. Moreover, the amino acid polymers/polypeptidesthat form random coil domain as disclosed herein are thought to bebiologically largely inert, especially with respect to proteolysis inblood plasma, immunogenicity, isoelectric point/electrostatic behaviour,binding to cell surface receptors as well as internalisation, but stillbiodegradable, which provides clear advantages over synthetic polymerssuch as PEG.

In accordance with the above, the present invention relates to abiologically active protein comprising the herein described biosyntheticrandom coil polypeptide. Such a biologically active protein/proteinconstruct comprising the biosynthetic random coil polypeptide decribedherein is a heterologous biological active protein/protein construct. Inparticular, herein disclosed is/are also (a) biologically active,heterologous protein(s) comprising or consisting of at least two domainswherein

-   -   (a) a first domain of said at least two domains comprises or        consists of an amino acid sequence having and/or mediating said        biological activity; and    -   (b) a second domain of said at least two domains comprises or        consists of the herein described and defined random coil        polypeptide or polypeptide segment.

It is of note that in accordance with the present invention that “firstdomain” and said “second domain” relate to protein stretches that arenot naturally occurring within the same protein or that are not expectedto be part of the same hypothetical protein as encoded by a codingnucleic acid sequence (like an open reading frame) as found in nature.

The definitions and explanations given herein above in context of therandom coil polypeptide or polypeptide segment thereof apply, mutatismutandis, in the context of biologically active proteins comprising saidrandom coil polypeptide (or (a) polypeptide segment(s) thereof).

Preferably, said random coil conformation mediates an increased in vivoand/or in vitro stability of said biologically active protein, like thein vivo and/or in vitro stability in biological samples or inphysiological environments.

For example, it is envisaged herein that proteins comprising a hereindefined, additional “second domain” adopting a random coil conformationin aqueous solution or under physiological conditions (for examplepolymers consisting of about 200 or about 400 or about 600 amino acidresidues and comprising PA#1/SEQ ID NO. 1, PA#2/SEQ ID NO. 2, PA#3/SEQID NO. 3, PA#4/SEQ ID NO. 4, PA#5/SEQ ID NO. 5, PA#6/SEQ ID NO. 6 and/orP1A1/SEQ ID NO. 51 as “building blocks”) have an advantageous serumstability or plasma half-life, even in vivo, (in particular ifintravenously administered) as compared to a control lacking said randomcoil conformation.

In WO 2008/155134 (as discussed herein above) it has been shown thatbiologically active proteins which comprise a domain with an amino acidsequence adopting a random coil conformation have an increased in vivoand/or in vitro stability. The random coil domains disclosed in WO2008/155134 consist, in particular, of proline, alanine, and serine(PAS) residues. The presence of these three residues is described inthis prior art document as an essential requirement for the formation ofa stable and soluble random coil in aqueous solution.

As discussed in the introduction herein above, WO 2007/103515 describesunstructured recombinant polymers which comprise as main constituents alarge variety of amino acids, inter alia, glycine, aspartate, alanine,serine, threonine, glutamate and proline. However, the term“unstructured recombinant polymer” has, in contrast to the terms“biosynthetic” and “random coil”, no recognized, clear meaning.

Also mentioned herein above was WO 2006/081249. This document describesprotein conjugates comprising a biologically active protein coupled to apolypeptide comprising 2 to 500 units of an amino acid repeat havingGly, Asn, and Gln as a major constituent and Ser, Thr, Asp, Gln, Glu,His, and Asn as a minor constituent. Said protein conjugates aredescribed to have either an increased or a decreased plasma half-lifewhen compared to the unconjugated biologically active protein. WO2006/081249, however, does not provide any teaching to predict whether aspecific amino acid repeat reduces or augments the plasma half-life ofthe conjugate. Moreover, WO 2006/081249 does not teach or suggest thatthe plasma half-life of proteins can be increased when the conjugatedprotein comprises an amino acid repeat that forms random coilconformation as shown in the present invention. Furthermore, the aminoacid repeats disclosed in WO 2006/081249 comprise at least two residuesselected from Gly, Asn, and Gln, which is in clear contrast with thebiosynthetic random coil polypeptides of the present invention whichcomprise an amino acid sequence that solely consists of proline andalanine amino acid residues.

Surprisingly, it has been found herein that biosynthetic random coilamino acid sequences as provided herein which, in contrast to the priorart, solely comprise proline and alanine residues (i.e. which preferablydo not comprise a substantial amount of any other amino acid, also not asubstantial amount of serine or no serine at all) do also form a usefulrandom coil structure. This is particularly unexpected given thedisclosure in WO 2008/155134 of fusion proteins with a domain composedonly of serine and alanine (SA) residues, i.e. where proline residueswere omitted, demonstrating that such a domain comprising only two typesof amino acids did not form a random coil, but a β-sheet structure.These serine-alanine domains did also not show such an increasedhydrodynamic volume as observed with “PAS” or, in particular, with the“P/A” sequences as provided herein.

As used herein, the term “biological activity” describes the biologicaleffect of a substance on living matter. Accordingly, the terms“biologically active protein” as used herein relate to proteins that arecapable of inducing a biological effect in living cells/organisms thatare exposed to said protein or polypeptide. Yet, it is of note that inthe context of the present invention, the term “biologically activeprotein” relates to the whole protein of the invention which bothcomprises an amino acid sequence having and/or mediating said biologicalactivity (said first domain) and the inventive amino acid sequenceadopting/forming random coil conformation and consisting solely ofproline and alanine (said second domain).

Accordingly, the terms “amino acid sequence having and/or mediatingbiological activity” or “amino acid sequence with biological activity”as used herein to the above-defined “first domain” of the biologicallyactive protein of the invention, which mediates or has or is capable ofmediating or having the above defined “biological activity”. Alsocomprised in the terms “amino acid sequence having and/or mediatingbiological activity” or “amino acid sequence with biological activity”are any proteins of interest (and functional fragments thereof, such asantibody fragments, fragments comprising extracellular or intracellulardomain(s) of a membrane receptor, truncated forms of a growth factor orcytokine and the like), the half-life of which, either in vivo or invitro, needs to be prolonged. In one embodiment of this invention, theamino acid sequence having and/or mediating biological activity inaccordance with the present invention may be deduced from any “proteinof interest”, i.e. any protein of pharmaceutical or biological interestor any protein that is useful as a therapeutic/diagnostic agent.

Accordingly, the biologically active proteins may comprise a firstdomain comprising a biologically active amino acid sequence which isderived from naturally produced polypeptides or polypeptides produced byrecombinant DNA technology. In a preferred embodiment, the protein ofinterest may be selected from the group consisting of bindingproteins/binding molecules, immunoglobulins, antibody fragments,transport proteins, membrane receptors, signaling proteins/peptides suchas cytokines, growth factors, hormones or enzymes and the like.

As explained herein above, the random coil polypeptide (or polypeptidesegment) comprised in the second domain of the biologically activeprotein forms the random coil conformation in particular underphysiological conditions. This is particularly relevant in context ofbiologically active proteins which may form part of a pharmaceuticalcomposition that is to be administered to a subject or patient.

It is of note that the inventive biosynthetic random coil domain (said“second domain”) of the biologically active protein natively (ie. underphysiologic conditions) adopts/forms/has random coil conformation, inparticular in vivo and when administered to mammals or human patients inneed of medical intervention. In contrast, it is known in the art thatproteins having a non-random secondary and/or tertiary structure asnative conformation tend to adopt a random coil conformation undernon-physiological conditions (i.e. under denaturing conditions).However, such denatured proteins have completely differentcharacteristics compared to the biologically active protein comprisingthe random coil polypeptide of the present invention. Hence, it is thegist of this invention that the “biologically active protein” and thebiologically active part of the fusion proteins/fusion constructs asprovided herein maintain their biological function also when combinedand/or linked with the biosynthetic random coil polypeptide (orpolypeptide segment) of this invention.

Furthermore, the random coil polypeptide (or polypeptide segment)retains solubility under physiological conditions. Accordingly, it isalso envisaged that the protein construct of the present invention(comprising the above defined “first” and “second domain”) may comprisethe “second”, random coil forming/adopting domain transiently ortemporarily not in random coil conformation, for example, when in formof a specific composition, like a lyophylisate or dried composition.Yet, it is important that such a “second domain” of the inventiveprotein construct again adopts after, e.g., reconstitution incorresponding buffers (preferably “physiological” buffers/excipientsand/or solvents), the herein defined random coil conformation. Said“second domain” is (if necessary, after corresponding reconstitution)capable of mediating an increased in vivo and/or in vitro stability ofthe inventive biologically active protein. It is preferred herein thatthe “second domain” as defined herein consists of the random coilpolypeptide (or polypeptide segment) of the present invention.

As used herein, the term “domain” relates to any region/part of an aminoacid sequence that is capable of autonomously adopting a specificstructure and/or function. In the context of the present invention,accordingly, a “domain” may represent a functional domain or astructural domain. As described herein, the proteins of the presentinvention comprise at least one domain/part having and/or mediatingbiological activity and at least one domain/part forming random coilconformation. Yet, the proteins of the invention also may consist ofmore than two domains and may comprise e.g. an additional linker orspacer structure between the herein defined two domains/parts or anotherdomain/part like, e.g. a protease sensitive cleavage site, an affinitytag such as the His₆-tag or the Strep-tag, a signal peptide, retentionpeptide, a targeting peptide like a membrane translocation peptide oradditional effector domains like antibody fragments for tumour targetingassociated with an anti-tumour toxin or an enzyme for prodrug-activationetc.

In another embodiment, the biologically active protein of the inventionhas a hydrodynamic volume as determined by analytical gel filtration(also known as size exclusion chromatography, SEC) of at least 50 kDa,preferably of at least 70 kDa, more preferably of at least 80 kDa, evenmore preferably of at least 100 kDa, particularly preferably of at least125 kDa and most preferably of at least 150 kDa. The person skilled inthe art is readily capable of determining the hydrodynamic volume ofspecific proteins. Exemplary methods have been described herein above incontext of the random coil polypeptide. A skilled person is easily inthe position to adapt such methods also in context of the biologicallyactive protein of the present invention. As described herein below, thehydrodynamic volume of the biologically active proteins of the inventionthat comprise the above defined second domain, i.e. the domaincomprising or consisting of the herein provided random coil polypeptide(or segment thereof) are shown to have an unexpectedly largehydrodynamic volume in relation to the estimated hydrodynamic volume fora corresponding folded, globular protein based on their molecular weightor number/composition of amino acid residues.

It should be noted that the first domain comprising an “amino acidsequence having and/or mediating biological activity” may also adopt itsbiological activity in the context of or after association with anotherpolypeptide or amino acid sequence. For example, the Fab fragment of anantibody such as the one of the anti-tumour antibody Herceptin(Eigenbrot (1993) J. Mol. Biol. 229:969-995) consists of two differentpolypeptide chain, the immunoglobulin light chain and a fragment of theimmunoglobulin heavy chain, which may furthermore be linked via (an)interchain disulfide bond(s). According to the present invention, it maybe sufficient to link one of those chains (e.g. via gene fusion) to therandom coil polypeptide (or polypeptide segment) while the fullbiologically active protein is reconstituted by means of associationwith the other chain. Such reconstitution may be achieved, for example,by co-expression of the different polypeptides (on the one hand a fusionprotein of one chain and the random coil polypeptide, on the other handthe other chain) in the same host cell, as described in the appendedexamples, or by reconstitution in vitro, for example, as part of arefolding protocol.

Accordingly, also such proteins (comprising tow separate polypeptidechains) are considered as biologically active proteins in accordancewith the present invention. In such a case, the first domain as definedherein may comprise two separate polypeptide chains which are linkedonly non-covalently. Furthermore, the independent chains of thebiologically active protein/domain may each be linked to the random coilpolypeptide (or polypeptide segment). Beside antibody fragments thereare many other homo- or hetero-oligomeric proteins of interest (forexample, insulin, hemoglobin and the like) that are composed of severalassociated polypeptide chains and which are subject to this invention.

As used herein, the term “binding protein” relates to a molecule that isable to specifically interact with (a) potential binding partner(s) sothat it is able to discriminate between said potential bindingpartner(s) and a plurality of different molecules as said potentialbinding partner(s) to such an extent that, from a pool of said pluralityof different molecules as potential binding partner(s), only saidpotential binding partner(s) is/are bound, or is/are significantlybound. Methods for the measurement of binding between a binding proteinand a potential binding partner are known in the art and can beroutinely performed, e.g., by using ELISA, isothermal titrationcalorimetry, equilibrium dialysis, pull down assays, surface plasmonresonance or a Biacore apparatus. Exemplary binding proteins/bindingmolecules which are useful in the context of the present inventioninclude, but are not limited to antibodies, antibody fragments such asFab fragments, F(ab′)₂ fragments, single chain variable fragments(scFv), isolated variable regions of antibodies (VL and/or VH regions),CDRs, single domain antibodies/immunoglobulins, CDR-derivedpeptidomimetics, lectins, immunoglobulin domains, fibronectin domains,protein A domains, SH3 domains, ankyrin repeat domains, lipocalins orvarious types of scaffold-derived binding proteins as described, forexample, in Skerra (2000) J Mol Recognit 13:167-187, Gebauer (2009) CurrOpin Chem Biol 13:245-255, Binz (2005) Nat Biotechnol 23:1257-1268 orNelson (2009) Nat Biotechnol 27:331-337.

Other exemplary biologically active proteins of interest (in particularproteins comprised in the first domain or constituting/being the firstdomain of the biologically active protein) which are useful in thecontext of the present invention include, but are not limited to,granulocyte colony stimulating factor, human growth hormone,alpha-interferon, beta-interferon, gamma-interferon, lambda-interferone,tumor necrosis factor, erythropoietin, coagulation factors such ascoagulation factor VIII, coagulation factor VIIa, coagulation factor IX,gp120/gp160, soluble tumor necrosis factor I and II receptor,thrombolytics such as reteplase, peptides with metabolic effects such asGLP-1 or exendin-4, immunosuppressive/immunoregulatory proteins likeinterleukin-1 receptor antagonists or anakinra, interleukin-2 andneutrophil gelatinase-associated lipocalin or other natural orengineered lipocalins or those proteins or compounds listed, forexample, in Walsh (2003) Nat Biotechnol 21:865-870 or Walsh (2004) Eur JPharm Biopharm 58:185-196 or listed in online databases such asbiopharma.com/approvals.html or drugbank.ca. Further biologically activeproteins (in particular proteins comprised in the first domain orconstituting/being the first domain of the biologically active protein)which may be employed in context of the present invention are, interalia, follicle-stimulating hormone, glucocerebrosidase, thymosin alpha1, glucagon, somatostatin, adenosine deaminase, interleukin 11,hematide, leptin, interleukin-20, interleukin-22 receptor subunit alpha(IL-22ra), interleukin-22, hyaluronidase, fibroblast growth factor 18,fibroblast growth factor 21, glucagon-like peptide 1, osteoprotegerin,IL-18 binding protein, growth hormone releasing factor, soluble TACIreceptor, thrombospondin-1, soluble VEGF receptor Flt-1, α-galactosidaseA, myostatin antagonist, gastric inhibitory polypeptide, alpha-1antitrypsin, IL-4 mutein, and the like. As will be evident from thedisclosure herein, the present invention also relates to comprising thebiosynthetic random coil proline/alanine polypeptide or proline/alaninepolypeptide segment and pharmaceutically or medically useful molecules,like small molecules, peptides or biomacromolecules such as proteins,nucleic acids, carbohydrates, lipid vesicles and the like, in particularpharmaceutically or medically useful proteins, like (but not limited to)binding proteins/binding molecules, immunoglobulins, antibody fragments,transport proteins, membrane receptors, signaling proteins/peptides,cytokines, growth factors, hormones or enzymes and the like may becomprised in the herein defined drug constructs btu they may also bepart of the herein defined biologically active, heterologous proteincomprising or consisting of said defined at least two domains. In such acase, said particular pharmaceutically or medically useful proteins (orfunctional fragments thereof) may be the “first domain” of said at leasttwo domains comprising or consisting of an amino acid sequence havingand/or mediating said biological activity Functional fragments, in thiscontext, are fragments of said pharmaceutically or medically usefulproteins that are still capable to elucidate the desired biological orpharmaceutical response in vivo and/or in vitro and/or still have ormediate the desired biological activity.

The above-mentioned polypeptide linker/spacer, inserted between saidfirst and said second domains, preferably comprises plural hydrophilic,peptide-bonded amino acids that are covalently linked to both domains.In yet another embodiment said polypeptide linker/spacer comprises aplasma protease cleavage site which allows the controlled release ofsaid first domain comprising a polypeptide having and/or mediating abiological activity. Linkers of different types or lengths may beidentified without undue burden to obtain optimal biological activity ofspecific proteins.

In a preferred embodiment, the biologically active proteins of thepresent invention are fusion proteins. A fusion protein as describedherein may comprise at least one domain which can mediate a biologicalactivity and at least one other domain which comprises the biosyntheticrandom coil polypeptide (or polypeptide segment) as described herein ina single multi-domain polypeptide. Again, it is of note that the presentinvention is not limited to fusion proteins wherein one domain mediatesa biological activity. Also other “fusion proteins”/“fusion constructs”are provided herein wherein one part/domain is or comprises theinventive random coil polypeptide/polymer of proline/alanine and theother part/domain comprises another protein stretch/structure.

In particular, in the case of fusion proteins the random coilpolypeptide (or polypeptide segment) according to this invention doesnot necessarily carry Pro or Ala residues at its amino or carboxylterminus. In an alternative embodiment, the biologically active proteinin accordance with the present invention may represent a proteinconjugate wherein a protein of interest or a polypeptide/polypeptidestretch/peptide/amino acid sequence having and/or mediating biologicalactivity is conjugated via a non-peptide bond to an amino acid sequencewhich forms/adopts random coil conformation, in particular, the randomcoil polypeptide (or polypeptide segment) as provided herein andconsisting solely of proline and alanine residues. Non-peptide bondsthat are useful for cross-linking proteins are known in the art with thebiosynthetic random coil polypeptide or polypeptide segment comprisingan amino acid sequence consisting solely of proline and alanine aminoacid residues, wherein said amino acid sequence consists of at least 50proline (Pro) and alanine (Ala) amino acid residues as provided herein.Such Non-peptide bonds may include disulfide bonds, e.g. between Cysside chains, thioether bonds or non-peptide covalent bonds induced bychemical cross-linkers, such as disuccinimidyl suberate (DSS) orsulfosuccinimidyl 4-[p-maleimidophenyl] butyrate (Sulfo-SMPB),metal-chelating/complexing groups, as well as non-covalentprotein-protein interactions.

It is of note that the “biologically active protein” of the presentinvention may also comprise more than one “amino acid sequence havingand/or mediating a biological activity”. Furthermore, the biologicallyactive protein may also comprise more than biosynthetic random coilpolypeptide (or segment thereof). In the simplest case, the biologicallyactive protein consists of two domains, i.e. a first domain comprisingan amino acid sequence having and/or mediating a biological activity anda second domain comprising the biosynthetic polypeptide (or segmentthereof). It is of note that the present invention is not limited to“biologically or therapeutically active proteins” linked to the hereindisclosed biosynthetic random coil polypeptide or polypeptide segmentcomprising an amino acid sequence consisting solely of proline andalanine amino acid residues, wherein said amino acid sequence consistsof at least 50 proline (Pro) and alanine (Ala) amino acid residues. Alsoother proteins or molecules of interest, as relevant for otherindustries, like food or beverage industry, cosmetic industry and thelike, may be manufactured by the means and methods provided herein.

The person skilled in the art is aware that the “domain comprising anamino acid sequence having and/or mediating a biological activity” andthe “second domain comprising the random coil polypeptide (or segmentthereof) as comprised in the biologically active proteins of theinvention may be organized in a specific order.

Accordingly, and in the context of the invention, the order of theherein defined “first” and “second” domain of the inventive biologicallyactive polypeptide may be arranged in an order, whereby said “firstdomain” (i.e. protein of interest; “amino acid sequence having and/ormediating said biological activity”) is located at the amino (N—)terminus and said “second domain” (i.e. the domain that comprises theherein provided random coil polypeptide (or segment thereof)) is locatedat the carboxy (C—) terminus of the biologically active protein.However, this order may also be reversed, e.g. said “first domain” (i.e.protein of interest; “amino acid sequence having and/or mediating saidbiological activity”) is located at the carboxy (C—) terminus and said“second domain” (i.e. the domain that comprises the herein providedrandom coil polypeptide (or segment thereof)) is located at the amino(N—) terminus of the biologically active protein. If the biologicallyactive protein consists only of one first domain and one second domain,the domain order may, accordingly, be (from N-terminus to C-terminus):first domain (amino acid sequence having and/or mediating a biologicalactivity)—second domain (random coil polypeptide (or segment thereof)).Vice versa, the domain order may be (from N-terminus to C-terminus):second domain (random coil polypeptide (or segment thereof))—firstdomain (amino acid sequence having and/or mediating a biologicalactivity).

It is also envisaged that more than one domain comprising or consistingof an amino acid sequence having and/or mediating said biologicalactivity are to be used in context of the inventive protein construct.For example, the biologically active protein may comprise two “firstdomain”, i.e. two specific amino acid sequences having and/or mediatinga biological activity, whereby this biological activity may be the sameor a different activity. If the biologically active protein consists oftwo such “first domains”, i.e two specific amino acid sequences havingand/or mediating a biological activity, and one “second domain”(comprising the biosynthetic random coil polypeptide (or segmentthereof), the domain order may be (from N-terminus to C-terminus): firstdomain (amino acid sequence having and/or mediating a specificbiological activity)—second domain (random coil polypeptide (or segmentthereof))—first domain (amino acid sequence having and/or mediating aspecific (optionally different) biological activity).

The same explanations apply in cases where the biologically activeprotein comprises more than one “second domain” (i.e. the biologicallyactive protein comprises more than one random coil polypeptide (orsegment thereof). If the biologically active protein consists of twosuch “second domains”, i.e two domains comprising the biosyntheticrandom coil polypeptide (or segment thereof), and one “first domain”(comprising an amino acid sequence having and/or mediating a biologicalactivity), the domain order may be (from N-terminus to C-terminus):second domain (random coil polypeptide (or segment thereof))—firstdomain (amino acid sequence having and/or mediating a specificbiological activity)—second domain (random coil polypeptide (or segmentthereof)). If the biologically active protein comprises more than one“second domain” it is envisaged herein that these “second domains” maybe identical or may be different.

As mentioned above, the biologically active protein may comprise morethan one “first domain”, i.e. more than one specific amino acidsequences having and/or mediating a biological activity and more thanone “second domain” (biosynthetic random coil polypeptide (or segmentthereof)) whereby these “first domains” may be identical or differentand/or whereby said “second domains” may be identical or different. Insuch cases the following, exemplary domain orders are conceivable (fromN-terminus to C-terminus):

-   -   first domain (amino acid sequence having and/or mediating a        specific biological activity)—second domain (random coil        polypeptide (or segment thereof))—first domain (amino acid        sequence having and/or mediating a specific biological        activity)—second domain (random coil polypeptide (or segment        thereof));    -   second domain (random coil polypeptide (or segment        thereof))—first domain (amino acid sequence having and/or        mediating a specific biological activity)—first domain (amino        acid sequence having and/or mediating a specific biological        activity)—second domain (random coil polypeptide (or segment        thereof));    -   first domain (amino acid sequence having and/or mediating a        specific biological activity)—second domain (random coil        polypeptide (or segment thereof))—second domain (random coil        polypeptide (or segment thereof))—first domain (amino acid        sequence having and/or mediating a specific biological        activity);    -   second domain (random coil polypeptide (or segment        thereof))—first domain (amino acid sequence having and/or        mediating a specific biological activity)—second domain (random        coil polypeptide (or segment thereof))—first domain (amino acid        sequence having and/or mediating a specific biological        activity);    -   second domain (random coil polypeptide (or segment        thereof))—second domain (random coil polypeptide (or segment        thereof))—first domain (amino acid sequence having and/or        mediating a specific biological activity)—first domain (amino        acid sequence having and/or mediating a specific biological        activity); or    -   first domain (amino acid sequence having and/or mediating a        specific biological activity)—first domain (amino acid sequence        having and/or mediating a specific biological activity)—second        domain (random coil polypeptide (or segment thereof))—second        domain (random coil polypeptide (or segment thereof)).

For a person skilled in the art further corresponding domain orders (inparticular in cases where more than two “first domains” or more than two“second domains” are comprised in the biologically active protein) areeasily conceivable.

As with all embodiments of the present inventivepolypeptide/biologically active protein, said domain(s) comprising anamino acid sequence having and/or mediating the said biological activitymay also be a biologically active fragment of a given protein with adesired biological function. Therefore, the herein defined “seconddomain” (preferably comprising the herein provided random coilpolypeptide (or segment thereof)) may also be located between twobiologically active fragments of a protein of interest or betweenbiologically active fragments of two proteins of interest. All theexplanations and definitions given herein above in context of “fulllength” proteins/polypeptides of interest (i.e. when the amino acidsequences has/mediates a certain biological activity on its own) apply,mutatis mutandis, in context of such fragments.

Again the above invention is not limited to the constructs that comprisea “domain” with a “biological active function”. The constructs of thepresent invention may also comprise domains with other functions and arenot limited to biological activities. These are merely embodiments ofthe present invention and it is evident for the skilled artisan thatother constructs can easly be made and used without deferring from thegist of the present invention. Accordingyl, the herein said in contextof “amino acid sequence having and/or mediating a specific biologicalactivity” applies, mutatis mutnatis, for other constructs, for exampleconstructs to be used in other technical fields, like in cosmetics, foodprocessing, dairy products, paper production, etc. As mentioned hereinabove, the biosynthetic polypeptides/polymers of the rpesent inventioncan also be used to be linked with e.g. small molecules and the like.

Again, it has to be pointed out that the term “amino acid sequencehaving and/or mediating first biological activity” is not limited tofull-length polypeptides that have and/or mediate said biologicalactivity or function, but also to biologically and/or pharmacologicallyactive fragments thereof. Especially, but not only, in a context whereintwo or more “first domains” as defined herein are comprised in theinventive “biologically active protein” it is also envisaged that these“first domains” are or represent different parts of a protein complex orfragments of such parts of protein complex.

As exemplified herein below, the biologically active proteins of theinvention which are modified to comprise a random coil polypeptidesurprisingly exhibit an increased in vivo and/or in vitro stability whencompared to unmodified biologically active proteins that lack saidrandom coil domain. As used herein, the term “in vivo stability” relatesto the capacity of a specific substance that is administered to theliving body to remain biologically available and biologically active. Invivo, a substance may be removed and/or inactivated due to excretion,kidney filtration, liver uptake, aggregation, degradation and/or othermetabolic processes. Accordingly, in the context of the presentinvention biologically active proteins that have an increased in vivostability may be less rapidly excreted through the kidneys (urine) orvia the feces and/or may be more stable against proteolysis, inparticular against in vivo proteolysis in biological fluids, like blood,liquor cerebrospinalis, peritoneal fluid, and lymph. In one embodiment,the increased in vivo stability of a biologically active proteinmanifests in a prolonged plasma half-life of said biologically activeprotein. In particular, the increased in vivo stability of thebiologically active protein is a prolonged plasma half-life of saidbiologically active protein comprising said second domain when comparedto the biologically active protein lacking the second domain.

Methods for measuring the in vivo stability of biologically activeproteins are known in the art. As exemplified herein below, biologicallyactive proteins may be specifically detected in the blood plasma usingWestern blotting techniques or enzyme linked immunosorbent assay(ELISA). Yet, the person skilled in the art is aware that other methodsmay be employed to specifically measure the plasma half-life of aprotein of interest. Such methods include, but are not limited to thephysical detection of a radioactively labelled protein of interest.Methods for radioactive labelling of proteins e.g. by radioiodinationare known in the art.

The term “increased in vitro stability” as used herein relates to thecapacity of a biologically active protein to resist degradation and/oraggregation and to maintain its original biological activity in an invitro environment. Methods for measuring the biological activity ofbiologically active proteins are well known in the art.

Furthermore, a drug conjugate is provided which comprises the hereindescribed and defined random coil polypeptide or polypeptide segment anda small molecule drug that is conjugated to said random coil polypeptideor polypeptide segment. Non-limiting examples of the small molecules aredigoxigenin, fluorescein doxorubicin, calicheamicin, camptothecin,fumagillin, dexamethasone, geldanamycin, paclitaxel, docetaxel,irinotecan, cyclosporine, buprenorphine, naltrexone, naloxone,vindesine, vancomycin, risperidone, aripiprazole, palonosetron,granisetron, cytarabine, NX1838, leuprolide, goserelin, buserelin,octreotide, teduglutide, cilengitide, abarelix, enfuvirtide, ghrelin andderivatives, tubulysins, platin derivatives, alpha 4 integrininhibitors, antisense nucleic acids, small interference RNAs, microRNAs, steroids, DNA or RNA aptamers, peptides, peptidomimetics. Ingeneral, the present invention also relates to drug constructscomprising the herein defined random coil polypeptide or polypeptidesegment and in particular pharmaceutically or medically usefulmolecules, like small molecules, peptides or biomacromolecules such asproteins, nucleic acids, carbohydrates, lipid vesicles and the like. Inthe appended illustrative experimental part (see, e.g. Example 22) thesuccessful generation of constructs/conjugates of the present inventionare provided, also constructs wherein “small chemical molecules” havebeen conjugated to the herein disclosed random coil polypeptide.Therefore, the present Figures and experimental information in thecorresponding figure legends provide for illustrative examples, whereinthe herein disclosed drug conjugates comprise (i) a biosynthetic randomcoil polypeptide or polypeptide segment comprising an amino acidsequence consisting solely of proline and alanine amino acid residues,wherein said amino acid sequence consists of at least 50 proline (Pro)and alanine (Ala) amino acid residues, and (ii) a small molecule, whichis, as illustration, selected from digoxigenin and fluorescein. It is ofnote that these are not only academic examples. Fluorescein orfluorescein derivates are are commonly used as diagnostics, and medicalfluorescein solutions are sold under the trade names Fluoescite®,AK-FLUOR® or Fluress®. Such compounds can certainly profit from themeans and methods provided herein. Digoxigenin forms the steroid part ofdigoxin, a well known secondary plant metabolite with cardioactivefunction which furthermore contains three digitoxose sugars. Digoxin,and to a lesser extent the closely related compound digitoxin, arewidely used for the treatment of ventricular tachyarrhythmias andcongestive heart failure (Hauptman (1999) Circulation 99: 1265-1270).All cardioactive steroids are potent and highly specific inhibitors ofthe Na⁺/K⁺-ATPase located in the cellular plasma membrane, therebyexerting sympatholytic or positive inotropic effects.

The definitions and explanations given herein above in context of therandom coil polypeptide or polypeptide segment thereof apply, mutatismutandis, in context of drug conjugate comprising the random coilpolypeptide (or polypeptide segment thereof) and a drug selected fromthe group consisting of (a) a biologically active protein or apolypeptide that comprises or that is an amino acid sequence that has ormediates a biological activity and (b) a small molecule drug.

The amino acid polymer forming random coil conformation/the random coilpolypeptide (or segment thereof) as defined and provided herein can beconjugated to a small molecule/small molecule drug. By this means,plasma half-life and/or solubility of the small molecule/small moleculedrug may be increased, unspecific toxicity may be decreased, and theprolonged exposure of active drug to target cells or structures in thebody may result in enhanced pharmacodynamics.

A site-specific conjugation of the N-terminus of the random coilpolypeptide with an activated drug derivative, e.g. asN-hydroxysuccinimide (NHS) ester derivative (Hermanson (1996)Bioconjugate Techniques, Academic Press, San Diego, Calif.), ispossible. Generally, the N-terminal amino group can be chemicallycoupled with a wide variety of functional groups such as aldehydes andketones (to form Schiff bases, which may be reduced to amines usingsodium borohydride or sodium cyanoborohydride, for example) or toactivated carbonic acid derivatives (anhydrides, chlorides, esters andthe like, to form amides) or to other reactive chemicals such asisocyanates, isothiocyanates, sulfonly chlorides etc. Also, theN-terminus of the amino acid polymer/polypeptide can first be modifiedwith a suitable protective group, for example an acetyl group, a BOCgroup or an FMOC group (Jakubke (1996) Peptide. Spektrum AkdemischerVerlag, Heidelberg, Germany) Furthermore, the amino terminus may beprotected by a pyroglutamyl group, which can form from an encoded Glnamino acid residue preceding the Pro/Ala polypeptide or polypeptidesegment. After activation of the C-terminal carboxylate group, e.g.using the common reagents EDC(N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide) and NHS, site-specificcoupling to the C-terminus of the protected random coil polypeptide canbe achieved if the drug carries a free amino group, for example.

Alternatively, the N-terminus or the C-terminus of the amino acidpolymer forming random coil conformation/the random coil polypeptide canbe modified with a commercially available linker reagent providing amaleimide group, thus allowing chemical coupling to a thiol group aspart of the drug molecule. In this manner uniform drug conjugates can beeasily obtained. Similar techniques, which are well known in the art(Hermanson (1996) loc. cit.), can be used to couple the random coilpolypeptide to a peptide or even to a protein drug. Such peptides orproteins can easily be prepared carrying a Lys or Cys side chain, whichallows their in vitro coupling to the amino acid polymer forming randomcoil conformation via NHS ester or maleimide active groups. Generally,similar drug conjugates can be prepared with fusion proteins comprisingthe random coil polypeptide (or segment thereof). Yet, and asillustrated in the appended Examples and Figures the present inventionalso provides for the preparation of a random coil polypeptides or arandom coil polypeptide segments as comprised in the innovativeconjugates of the present invention.

As an alternative to a single site-specific conjugation the random coilpolypeptide may be equipped with additional side chains, at the N- orC-terminus or internally, suitable for chemical modification such aslysine residues with their ε-amino groups, cysteine residues with theirthiol groups, or even non-natural amino acids, allowing the conjugationof multiple small molecules using, for example, NHS ester or maleimideactive groups.

Apart from stable conjugation, a prodrug may be linked transiently tothe random coil polypeptide. The linkage can be designed to be cleavedin vivo, in a predictable fashion, either via an enzymatic mechanism orby slow hydrolysis initiated at physiological pH similarly as, forexample, the poorly soluble antitumor agent camptothecin was conjugatedto a PEG polymer, thus achieving increased biodistribution, decreasedtoxicity, enhanced efficacy and tumor accumulation (Conover (1998)Cancer Chemother Pharmacol, 42:407-414). Examples for further prodrugsare chemotherapeutic agents like docetaxel (Liu (2008) J Pharm Sci.97:3274-3290), doxorubicin (Veronese (2005) Bioconjugate Chem. 16:775-784) or paclitaxel (Greenwald (2001) J Control Release 74:159-171).

Furthermore, the small molecule may be coupled to a fusion proteincomprising the amino acid polymer/polypeptide genetically fused to atargeting domain, e.g. an antibody fragment, thus resulting in aspecific delivery of the small molecule drug. In the latter caseimmunotoxins can be easily generated by conjugation with a cytotoxicsmall molecule if the targeting domain is directed against acell-surface receptor which undergoes internalization, for example.

In accordance with the above, the present invention also relates,therefore, to the provision of the herein disclosed biosynthetic randomcoil polypeptide or polypeptide segment comprising an amino acidsequence consisting solely of proline and alanine amino acid residuesfor further and additional coupling with other compounds of choice. Saidfurther and/or additional coupling may be and/or may comprise the firstcoupling of said biosynthetic random coil polypeptide or biosyntheticrandom coil polypeptide segment with or to another compound.

In another embodiment, the present invention relates to nucleic acidmolecules encoding the random coil polypeptides (or segments thereof) orbiologically active proteins as described herein. Accordingly, saidnucleic acid molecule may comprise a nucleic acid sequence encoding apolypeptide having biological activity and a nucleic acid sequenceencoding the random coil polypeptide (or segment thereof) . . . . Theterm “nucleic acid molecule”, as used herein, is intended to includenucleic acid molecules such as DNA molecules and RNA molecules. Saidnucleic acid molecule may be single-stranded or double-stranded, butpreferably is double-stranded DNA. Preferably, said nucleic acidmolecule may be comprised in a vector.

Accordingly, the present invention also relates to a nucleic acidmolecule encoding the random coil polypeptide or polypeptide segment ascomprised in the conjugates provided herein, like a drug conjugate asdefined herein, or a nucleic acid molecule encoding a protein conjugatethat comprises a biologically active protein as defined above and thatcomprises, additionally, a biosynthetic random coil polypeptide orpolypeptide segment comprising an amino acid sequence consisting solelyof proline and alanine amino acid residues, wherein said amino acidsequence consists of at least 50 proline (Pro) and alanine (Ala) aminoacid residues.

In one embodiment a nucleic acid molecule is provided that encodes aconjugate, like a drug conjugate or food conjugate as defined above,said nucleic acid molecule comprising

-   -   (i) a nucleic acid sequence encoding a translated amino acid        and/or a leader sequence;    -   (ii) a nucleic acid sequence encoding a biosynthetic random coil        polypeptide or polypeptide segment comprising an amino acid        sequence consisting solely of proline and alanine amino acid        residues, wherein said amino acid sequence consists of at least        50 proline (Pro) and alanine (Ala) amino acid residues;    -   (iii) a nucleic acid sequence encoding biologically active        protein or said a polypeptide that comprises or that is an amino        acid sequence that has or that mediates a biological activity or        a protein of interest, like a protein to be employed in other        industrial areas like the food industry; and    -   (iv) a nucleic acid sequence that represents or is a        translational stop codon.

The above mentioned “translated amino acid and/or a leader sequence”under (i) may for example be the starting “M”, i.e. a methionine derivedfrom a corresponding starting codon, it may also comprise non-translatedsequences of an mRNA like the 5′ sequence up to a start codon whichcomprises for example a ribosome binding site. Such a sequence mayhowever also comprise classical leader and/or signal sequences forexample for secretion of an expressed protein into the periplasm or in aculture medium. Prokaryotic signal peptides are for example OmpA, MalE,PhoA, DsbA, pelB, Afa, npr, STII. Eukaryotic signal peptides are forexample Honeybee melittin signal sequence, acidic glycoprotein gp67signal sequence, mouse IgM signal sequence, hGH signal sequence.

Biologically active proteins or polypeptides that comprises or that isan amino acid sequence that has or that mediates a biological activityas well as other proteins of interest, like a protein to be employed inother industrial areas, have been provided herein above. Saidembodiments apply, mutatis mutantis, for the nucleic acid molece(part/segments (iii)) as illustrated herein above.

Translational stop codons to be employed in the nucleic acid moleculeprovided herein are well known in the art and are, e.g. codons UAA, UAGor UGA.

In one embodiment of the nucleic acid molecule as provided herein abovesaid nucleic acid molecule parts/segments (ii) and (iii) areinterchanged in their position on said nucleic acid molecule encodingfor a conjugate, like a drug conjugate or a food conjugate. Such anucleic acid molecule would comprise the following order ofparts/segments:

-   -   (i) a nucleic acid sequence encoding a translated amino acid        and/or a leader sequence;    -   (ii) a nucleic acid sequence encoding biologically active        protein or said a polypeptide that comprises or that is an amino        acid sequence that has or that mediates a biological activity or        a protein of interest, like a protein to be employed in other        industrial areas, like the food industry;    -   (iii) a nucleic acid sequence encoding a biosynthetic random        coil polypeptide or polypeptide segment comprising an amino acid        sequence consisting solely of proline and alanine amino acid        residues, wherein said amino acid sequence consists of at least        50 proline (Pro) and alanine (Ala) amino acid residues; and    -   (iv) a nucleic acid sequence that represents or is a        translational stop codon.

The nucleic acid molecules as provided herein above may also,optionally, comprise, between parts/segments (i) and (ii) and/or betweenparts/segments (ii) and (iii), a protease and/or a chemical cleavagesite and/or a recognition site. Such chemical cleavage sites are wellknown in the art, and comprise for example specific, individual aminoacid sequences (see, e.g. Lottspeich and Engels (Hrsg.) (2006)Bioanalytik. 2. Auflage. Spektrum Akademischer Verlag, Elsevier,Munchen, Germany). For example, cyanogen bromide or cyanogen chloridecleaves the peptide bond following a Met residue; hydroxylamine cleavesthe asparaginyl-glycyl bond; formic acid cleaves Asp-Pro;2-(2′-nitrophenylsulfenyl)-3-methyl-3-bromoinolenine, 2-iodosobenzoicacid or N-chlorosuccinimide after Trp; 2-nitro-5-thiocyanatobenzoic acidafter Cys. It is also envisaged and possible that the residue precedingthe Pro/Ala polypeptide or polypeptide segment may be substituted to Metvia site-directed mutagenesis and the resulting fusion protein can thenbe cleaved by BrCN. Similarly, other amino acid sequences comprisingcleavage site can be introduced into the recombinant fusion protein orits encoding nucleic acid by way of site-directed mutagenesis.

Also useful protease recognition/cleavage sites are known in the art.These comprise, but are not limited to: trypsin, chymotrypsin,enterokinase, Tobacco Etch Virus (TEV) protease, PreScission protease,HRV 3C Protease, SUMO Protease, Sortase A, granzyme B, furin, thrombin,factor Xa or self cleavable inteins. Factor Xa hydrolyses the peptidebond at the C-terminal end of the amino acid sequence IleGluGlyArg,which may be inserted between the N-terminal fusion partner and thePro/Ala polypeptide or polypeptide segment. A particularly simple methodto achieve proteolytic cleavage would be by insertion or substitution ofa Lys or Arg side chain at the N-terminal end of the Pro/Ala polypeptideor polypeptide segment followed by digest with trypsin, which does notcleave within the Pro/Ala polypeptide or polypeptide segment as long asinternal Lys or Arg side chains are avoided. Illustrative recognitionsites are, without being limited, D-D-D-D-K (enterokinase), ENLYFQ(G/S)(TEV protease), I-(E/D)-G-R (Factor Xa), L-E-V-L-F-Q-G-P (HRV 3C),R-X-(K/R)-R (Fuxin), LPXTG (Sortase A), L-V-P-R-G (Thrombin) orI-E-X-D-X-G (Granzyme B).

As is evident form the disclosure herein above, the present inventionprovides for recombinantly produced biosynthetic random coilpolypeptides and polypeptide segments that can be conjugated withmolecules of choice, like useful proteins, pharmaceutically activepolypeptides or small molecules, diagnostically useful polypeptides orsmall molecules or, inter alia, other useful proteins or small moleculesof other industrial areas, like food or paper industry or in oilrecovery. Therefore, the present invention also provide for a nucleicacid encoding for a biosynthetic random coil polypeptide or polypeptidesegment comprising an amino acid sequence consisting solely of prolineand alanine amino acid residues wherein said amino acid sequenceconsists of at least 50 proline (Pro) and alanine (Ala) amino acidresidues, said nucleic acid molecule comprising

-   -   (i) a nucleic acid sequence encoding for a translated amino acid        and/or leader sequence;    -   (ii) a nucleic acid sequence encoding for said a biosynthetic        random coil polypeptide or polypeptide segment comprising an        amino acid sequence consisting solely of proline and alanine        amino acid residues; and    -   (iii) a nucleic acid sequence that represents or is a        translational stop codon.

Such a nucleic acid molecule may, optionally, comprise, between (i) and(ii) a protease and/or a chemical cleavage site and/or a recognitionsite).

Also for this nucleic acid molecule, the embodiments provided hereinabove in context of the first two described nucleic acid molecules (i.e.a protease and/or a chemical cleavage site and/or a recognition), applyhere mutatis mutantis.

Useful and illustrative signal sequences to be employed in context ofthis invention comprise, but are not limited, prokaryotic sequenceslike: OmpA, MalE, PhoA, DsbA, pelB, Afa, npr, STII or eukaryoticsequences like: Honeybee melittin signal sequence, acidic glycoproteingp67 signal sequence, mouse IgM signal sequence, hGH signal sequence

In particular the nucleic acid molecule encoding the biosynthetic randomcoil polypeptide or polypeptide segment comprising an amino acidsequence consisting solely of proline and alanine amino acid residues ofthe present invention is useful in methods as also provided herein belowand as illustrated in the appended examples and figures. Such anexpressed random coil polypeptide or random coil polypeptide segment canbe isolated form, e.g. host cells expressing such a random coilpolypeptide or random coil polypeptide segment. Such host cells may betransfected cells, for example with an vector as provided herein.

Therefore, it is envisaged to transfect cells with the nucleic acidmolecule or vectors as described herein. In a further embodiment, thepresent invention relates to nucleic acid molecules which uponexpression encode the random coil polypeptide (or segment thereof) orbiologically active proteins of the invention. Yet, in a furtherembodiment, the present invention relates to nucleic acid moleculeswhich upon expression encode the herein disclosed polypeptides that,entirely or in part, form/adopt random coil conformation in aqueoussolution or under physiological conditions. Said nucleic acid moleculesmay be fused to suitable expression control sequences known in the artto ensure proper transcription and translation of the polypeptide aswell as signal sequences to ensure cellular secretion or targeting toorganelles. Such vectors may comprise further genes such as marker geneswhich allow for the selection of said vector in a suitable host cell andunder suitable conditions.

Preferably, the nucleic acid molecule of the invention is comprised in arecombinant vector in which a nucleic acid molecule encoding the hereindescribed biologically active protein is operatively linked toexpression control sequences allowing expression in prokaryotic oreukaryotic cells. Expression of said nucleic acid molecule comprisestranscription of the nucleic acid molecule into a translatable mRNA.Regulatory elements permitting expression in prokaryotic host cellscomprise, e.g., the lambda PL, lac, trp, tac, ara, phoA, tet or T7promoters in E. coli. Possible regulatory elements ensuring expressionin eukaryotic cells, preferably mammalian cells or yeast, are well knownto those skilled in the art. They usually comprise regulatory sequencesensuring initiation of transcription and optionally poly-A signalseffecting termination of transcription and stabilization of thetranscript. Additional regulatory elements may include transcriptionalas well as translational enhancers, and/or naturally associated orheterologous promoter regions. Examples for regulatory elementspermitting expression in eukaryotic host cells are the AOX1 or GAL1promoters in yeast or the CMV, SV40, RSV (Rous sarcoma virus) promoters,CMV enhancer, SV40 enhancer or a globin intron in mammalian and otheranimal cells. Apart from elements which are responsible for theinitiation of transcription such regulatory elements may also comprisetranscription termination signals, such as the SV40-poly-A site or thetk-poly-A site, downstream of the coding region.

Methods which are well known to those skilled in the art can be used toconstruct recombinant vectors (see, for example, the techniquesdescribed in Sambrook (1989), Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory NY and Ausubel (1989), Current Protocolsin Molecular Biology, Green Publishing Associates and WileyInterscience, NY). In this context, suitable expression vectors areknown in the art such as Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3, pP1CZalpha A (Invitrogen),or pSPORT1 (GIBCO BRL). Furthermore, depending on the expression systemthat is used, leader sequences capable of directing the polypeptide to acellular compartment or secreting it into the culture medium may beadded to the coding sequence of the nucleic acid molecule of theinvention.

The present invention also relates to vectors, particularly plasmids,cosmids, viruses, and bacteriophages that are conventionally employed ingenetic engineering, comprising a nucleic acid molecule encoding the therandom coil polypeptide (or segment thereof) or the biologically activeprotein of the invention. Preferably, said vector is an expressionvector and/or a gene transfer or targeting vector. Expression vectorsderived from viruses such as retroviruses, vaccinia virus,adeno-associated virus, herpes viruses or bovine papilloma virus may beused for delivery of the polynucleotides or vector of the invention intotargeted cell populations.

The vectors containing the nucleic acid molecules of the invention canbe transfected into the host cell by well known methods, which varydepending on the type of cell. Accordingly, the invention furtherrelates to a cell comprising said nucleic acid molecule or said vector.Such methods, for example, include the techniques described in Sambrook(1989), loc. cit. and Ausubel (1989), loc. cit. Accordingly, calciumchloride transfection or electroporation is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts (Sambrook (1989),loc. cit.). As a further alternative, the nucleic acid molecules andvectors of the invention can be reconstituted into liposomes fordelivery to target cells. The nucleic acid molecule or vector of theinvention which is present in the host cell may either be integratedinto the genome of the host cell or it may be maintainedextra-chromosomally. Accordingly, the present invention also relates toa host cell comprising the nucleic acid molecule and/or the vector ofthis invention. Host cells for the expression of polypeptides are wellknown in the art and comprise prokaryotic cells as well as eukaryoticcells, e.g. E. coli cells, yeast cells, invertebrate cells, CHO cells,CHO-K1 cells, HEK 293 cells, Hela cells, COS-1 monkey cells, melanomacells such as Bowes cells, mouse L-929 cells, 3T3 cell lines derivedfrom Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines and thelike.

In a further aspect, the present invention comprises methods for thepreparation of the conjugates of the present invention as well as thebiosynthetic random coil polypeptide (or segment thereof) orbiologically active proteins provided herein and comprising culturingthe (host) cell of this invention and isolating said random coilpolypeptide (or segment thereof) or the conjugate or a biologicallyactive protein from the culture as described herein. In general, theinventive random coil polypeptide (or segment thereof), the conjugate orbiologically active protein comprising a random coil domain may beproduced by recombinant DNA technology, e.g. by cultivating a cellcomprising the described nucleic acid molecule or vectors which encodethe inventive biologically active protein or random coil polypeptide (orsegment thereof) and isolating said protein/polypeptide from theculture. The inventive biologically active protein or random coilpolypeptide (or segment thereof) may be produced in any suitable cellculture system including prokaryotic cells, e.g. E. coli BL21, KS272 orJM83, or eukaryotic cells, e.g. Pichia pastoris, yeast strain X-33 orCHO cells. Further suitable cell lines known in the art are obtainablefrom cell line depositories like the American Type Culture Collection(ATCC).

The term “prokaryotic” is meant to include bacterial cells while theterm “eukaryotic” is meant to include yeast, higher plant, insect andmammalian cells. The transformed hosts can be grown in fermentors andcultured according to techniques known in the art to achieve optimalcell growth. In a further embodiment, the present invention relates to aprocess for the preparation of a random coil polypeptide (or segmentthereof) or a biologically active protein described above comprisingcultivating a cell of the invention under conditions suitable forexpression of the biologically active protein or random coil polypeptide(or segment thereof) and isolating said protein/polypeptide from thecell or the culture medium.

The random coil polypeptide (or segment thereof) per se of the presentinvention does, preferably not comprises any chemically reactive group,except for, possibly, one N-terminal primary (or, in the case ofproline, secondary) amino group and one carboxylate group at theC-terminus of the polymer. However, it is evident for the skilledartisan that the biosynthetic random coil polypeptide/polymer asprovided herein may comprise a chemically reactive group, for examplewhen said random coil polypeptide/polymer is part of a “fusionprotein”/“fusion construct”. As also described above, the biosyntheticrandom coil polypeptide (or segment thereof) can be prepared byrecombinant expression in a transformed cell in several ways accordingto methods well known to the person skilled in the art, for example: (i)direct expression in the cytoplasm with the help of an N-terminal Metresidue/start codon; (ii) secretion via an N-terminal signal peptide,for example OmpA, PhoA (Monteilhet (1993) Gene. 1993 125:223-228),mellitin (Tessier (1991) Gene 98: 177-183), interleukin 2 (Zhang (2005)J Gene Med 7: 354-365), hGH (Pecceu (1991) Gene 97(2):253-258) and thelike, followed by intracellular cleavage resulting in the matureN-terminus, such as Ala or Pro; (iii) expression as a fusion proteinwith another soluble protein, e.g., maltose-binding protein at theN-terminus and with a protease cleavage site interspersed (Kapust andWaugh (2000) Protein Expr. Purif. 19:312-318), followed by specificprotease cleavage in vitro or in vivo, thus releasing the amino acidpolymer/polypeptide with its mature N-terminus such, as Ala or Pro.Another suitable fusion partner is the SUMO protein, which can becleaved by SUMO protease, as described in Examples 20 and 21. Furtherfusion partners include, without limitation, glutathion-S-transferase,thioredoxin, a cellulose-binding domain, an albumin-binding domain, afluorescent protein (such as GFP), protein A, protein G, an intein andthe like (Malhotra (2009) Methods Enzymol. 463:239-258).

As explained above, the random coil polypeptides (or polypeptidesegments)/polymers described consist predominantly of alanine andproline residues, whereas serine, threonine or asparagine, which arerequired for O- or N-glycosylation, are preferably absent. Thus, theproduction of the polypeptide itself or of a biologically active proteincomprising the random coil polypeptide (or polypeptide segment thereof)or, generally, a fusion protein comprising the random coil polypeptide(or polypeptide segment thereof) surprisingly can result in amonodisperse product preferably devoid of post-translationalmodifications within the Pro-Ala sequence This is an advantage forrecombinant protein production in eukaryotic cells, like chinese hamsterovarian cells (CHO) or yeast, which are often chosen for thebiosynthesis of complex proteins. For example, yeast has been used forthe production of approved therapeutic proteins such as insulin,granulocyte-macrophage colony stimulating factor, platelet-derivedgrowth factor or hirudin (Gerngross (2004) Nat. Biotechnol.22:1409-1414). CHO cells have served for the production of therapeuticproteins such as coagulation factor IX, interferone β-1a, tenecteplase(Chu (2001) Curr. Opin. Biotechnol. 12:180-187) or gonadotropins, wherethe glycocomponent may positively influence several aspects likefunctional activity, folding, dimerization, secretion as well asreceptor interaction, signal transduction, and metabolic clearance(Walsh (2006) Nat. Biotechnol. 24:-1241-1252). Accordingly, thepreparation of the inventive constructs, random coil polypeptides andconjugates in eukaryotic expression systems is also disclosed in contextof the present invention.

With the means and methods provided herein it is now possible tomanufacture and provide for the herein disclosed conjugates andmolecules comprising (i) a biosynthetic random coil polypeptide orpolypeptide segment comprising an amino acid sequence consisting solelyof proline and alanine amino acid residues and (ii) a further moleculeof interest, like a useful protein, a protein segment or a smallmolecule. The present invention, therefore, also provides for methodsfor the preparation or manufacure of such conjugates as well as ofbiosynthetic random coil polypeptides and/or molecules or conjugatescomprising the same. Accordingly, the present invention also providesfor a method for the preparation and/or manufacture of a random coilpolypeptide or a random coil polypeptide segment as comprised in theconjugates, like drug conjugates, food conjugates, diagnostic conjugatesand the like. Also methods for the preparation and/or manufacture of thebiologically active protein or conjugate comprising the random coilpolypeptide or the random coil polypeptide segment are provided.Furthermore, methods for the preparation and/or manufacture and/or forthe preparation and/or manufacture of a polypeptide that comprises orthat is an amino acid sequence that has or that mediates a biologicalactivity and that additionally comprises said random coil polypeptide orrandom coil polypeptide segment are provided. These methods, inparticular comprise (as one step) the cultuvation of the (host) cell asprovided herein above and (as a further step) the isolation of saidrandom coil polypeptide or biologically active protein and/or saidbiologically active protein and/or said polypeptide conjugate from theculture or from said cell. This isolated random coil, a biosyntheticrandom coil polypeptide or polypeptide segment comprising an amino acidsequence consisting solely of proline and alanine amino acid residues aswell as the isolated conjugate may than be further proceesed. Forexample, said biosynthetic random coil polypeptide or polypeptidesegment comprising an amino acid sequence consisting solely of prolineand alanine amino acid residues may be chemically linked or coupled to amolecule of interest, as also shown in the appended examples.Furthermore and as an alternative, the molecule of interest may beenzymatically conjugated e.g. via transglutaminase (Besheer (2009) JPharm Sci. 98:4420-8) or other enzymes (Subul (2009) Org. Biomol. Chem.7:3361-3371) to the said biosynthetic random coil polypeptide orpolypeptide segment comprising an amino acid sequence consisting ofproline and alanine amino acid residues.

The random coil polypeptide (or segment thereof) and/or a proteinconjugate comprising random coil polypeptide (or segment thereof) and aprotein of interest, like a biologically or therapeutically activeprotein or a protein to be used in, e.g. diagnostic methods, can beisolated (inter alia) from the growth medium, cellular lysates,periplasm or cellular membrane fractions. (Again, the present inventionis not limited to (protein) conjugates that are useful in a medical orpharmaceutical setting. The means and methods provided herein are alsoof use in other industrial areas, like, but not limited to food andbeverage industry, nutrient industry, paper industry, bioreagentindustry, research tool and reagent industry, industries where enzymesare to be used, cosmetic industry, oil processing and oil recovery, andthe like). The isolation and purification of the expressed polypeptidesof the invention may be performed by any conventional means (Scopes(1982) “Protein Purification”, Springer, New York, N.Y.), includingammonium sulphate precipitation, affinity purification, columnchromatography, gel electrophoresis and the like and may involve the useof monoclonal or polyclonal antibodies directed, e.g., against a tagfused with the biologically active protein of the invention. Forexample, the protein can be purified via the Strep-tag II usingstreptavidin affinity chromatography (Skerra (2000) Methods Enzymol.326:271-304) as described in the appended examples. Substantially purepolypeptides of at least about 90 to 95% homogeneity (on the proteinlevel) are preferred, and 98 to 99% or more homogeneity are mostpreferred, in particular for pharmaceutical use/applications. Dependingupon the host cell/organism employed in the production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated.

The invention further relates to the use of the biologically activeprotein, the random coil polypeptide (or segment thereof) or theconjugates, like the drug conjugates of the invention, the nucleic acidmolecule of the invention, the vector of the invention or the (host)cell of the invention for the preparation of a medicament, wherein saidbiologically active protein or drug (or any other small molecule orprotein of interest) has an increased in vivo and/or in vitro stabilityas compared to a control molecule that does not comprise or that is notlinked to a biosynthetic random coil polypeptide or polypeptide segmentcomprising an amino acid sequence consisting solely of proline andalanine amino acid residues, wherein said amino acid sequence consistsof at least 50 proline (Pro) and alanine (Ala) amino acid residues.

In yet another embodiment, the present invention relates to a method forthe treatment of diseases and/or disorders that benefit from theimproved stability of said biologically active protein or drug,comprising administering the biologically active protein or drugconjugate as described herein to a mammal in need of such treatment.Depending on the biological activity of the inventive protein or drugconjugate, the skilled person is readily capable of determining whichdisease/disorder is to be treated with a specific biologically activeprotein or drug conjugate of the invention. Some non-limiting examplesare listed in the following Table:

Biologically active protein (or a biologically active component/fragmentthereof) or drug Disorder/disease to be treated granulocyte colonystimulating cancer and/or chemotherapy related neutropenia factor humangrowth hormone growth hormone deficiency related hypoglycaemia and/orgrowth failure interferon α cancer, viral infection, hepatitis Cinterferon β auto-immune disease, multiple sclerosis interferon γ viralinfection tumor necrosis factor cancer interleukin-20 psoriasisα-galactosidase A Fabry disease myostatin antagonist sarcopenia gastricinhibitory polypeptide type 2 diabetes alpha-1 antitrypsin enzymereplacement therapy, cystic fibrosis, chronic obstructive pulmonarydiseases, acute respiratory syndrome, severe asthma. erythropoietinanaemia coagulation factor VIII haemophilia gp120/gp160 HIV solubletumor necrosis factor I inflammatory disease and II receptor reteplasethrombosis, myocardial infarction exendin-4 diabetes interleukin-1receptor auto-immune disease, rheumatoid arthritis antagonist (IL-1ra;anakinra) interleukin-2 cancer insulin diabetes asparaginase acutelymphoblastic leukemia, non-Hodgkin's lymphoma onconase malignantmesothelioma and other types of cancer streptokinase thromboticdisorders neutrophil gelatinase- microbial infection, kidney reperfusioninjury associated lipocalin antibodies and their fragments,immunological, oncological, neovascular, and infectious including singledomain diseases etc. antibodies, single chain and other engineeredfragments including CDR mimetic peptides and CDRs granulocyte-macrophagechemotherapy related neutropenia colony-stimulating factorfollicle-stimulating hormone infertility glucocerebrosidase Gaucher'sdisease thymosin alpha 1 chronic hepatitis B, cancer glucagonhypoglycemia somatostatin acromegaly adenosine deaminase adenosinedeaminase deficiency Interleukin-11 thrombocytopenia coagulation factorVIIa haemophilia coagulation factor IX hemophilia hematide anemiainterferone λ hepatitis C leptin lipodystrophy, obesity, Alzheimer'sdisease, type I diabetes interleukin-22 receptor subunit psoriasis alpha(IL-22ra) interleukin 22 metastatic melanoma hyaluronidase solid tumorsfibroblast growth factor 18 osteoarthritis fibroblast growth factor 21diabetes type II, obesity, dyslipidemia, metabolic disordersglucagon-like peptide 1 diabetes osteoprotegerin cancer, osteoporosis,rheumatoid arthritis IL-18 binding protein rheumatoid arthritis growthhormone-releasing HIV-associated lipodystrophy factor soluble TACIreceptor systemic lupus erythematosus, multiple sclerosis, rheumatoidarthritis thrombospondin-1 cancer soluble VEGF receptor Flt-1 cancerIL-4 mutein (IL-4 receptor asthma antagonist) cyclosporine organrejection fumagillin cancer naltrexone alcohol dependence octreotideacromegaly, carcinoid tumors teduglutide short bowel syndrome, Crohn'sdisease goserelin advanced prostate cancer, breast cancer camptothecincancer vancomycin Gram-positive pneumonias

In accordance with the above, the biologically active protein, therandom coil polypeptide (or segment thereof), the drug conjugate, thenucleic acid, the vector or the cell may be used for the preparation ofa medicament which preferably has or confers an increased in vivo and/orin vitro stability, in particular for the biologically active proteinand/or drug component, for the treatment of hormone deficiencies orrelated disorders, auto-immune disease, cancer, anaemia, neovasculardiseases, infectious/inflammatory diseases, thrombosis, myocardialinfarction, diabetes, infertility, Gaucher's disease, hepatitis,hypoglycaemia, acromegaly, adenosine deaminase deficiency,thrombocytopenia, haemophilia, anemia, obesity, Alzheimer's disease,lipodistrophy, psoriasis, metastatic melanoma, osteoarthritis,dyslipidemia, rheumatoid arthritis, systemic lupus erythromatosis,multiple sclerosis, asthma, osteoporosis, and reperfusion injury orother kidney diseases, for example. In one embodiment, the biologicallyactive protein, the drug conjugate the nucleic acid, the vector or thecell is for the use as a medicament which has an increased in vivoand/or in vitro stability of said biologically active protein/drugconjugate. Similarly, the biologically active protein, the random coilpolypeptide (or segment thereof), the drug conjugate, the nucleic acid,the vector or the cell are for use in the treatment of for the treatmentof hormone deficiencies or related disorders, auto-immune disease,proliferative disorders, like cancer, anaemia, neovascular diseases,infectious and/or inflammatory diseases, thrombosis, myocardialinfarction stroke, diabetes, infertility, penile dysfunction, Gaucher'sdisease, Fabry disease, sarcopenia, cystic fibrosis, obstructivepulmonary diseases, acute respiratory syndrome, hepatitis,hypoglycaemia, acromegaly, adenosine deaminase deficiency,thrombocytopenia, haemophilia, anemia, obesity, Alzheimer's disease,lipodistrophy, psoriasis, metastatic melanoma, osteoarthritis,dyslipidemia, rheumatoid arthritis, systemic lupus erythromatosis,multiple sclerosis, asthma, osteoporosis, and reperfusion injury orother kidney diseases, for example.

The present invention also relates to the use of the nucleic acidmolecules, vectors as well as transfected cells as provided herein andcomprising the nucleic acid molecules or vectors of the presentinvention in medical approaches, like, e.g. cell based gene therapyapproaches or nucleic acid based gene therapy approaches.

In a further embodiment, the random coil polypeptide (or polypeptidesegment thereof) as provided herein, the biologically active,heterologous protein/protein construct or the drug or food conjugate orother conjugates that comprise the biosynthetic random coil polypeptide(or polypeptide segment thereof) and/or the nucleic acid molecule or thevector or the host cell of the present invention) is part of acomposition. Said composition may comprise one or more of the inventiverandom coil polypeptides (or polypeptide segments thereof), biologicallyactive proteins, food conjugates, conjugates of interest, drugconjugates or nucleic acid molecules, vectors and/or host cells encodingand/or expressing the same. Said composition may be a pharmaceuticalcomposition, optionally further comprising a pharmaceutically acceptablecarrier and/or diluent. In a further embodiment, the present inventionrelates to the use of the herein described biologically active protein,the random coil polypeptide (or segment thereof) or the drug conjugatefor the preparation of a pharmaceutical composition for the prevention,treatment or amelioration of diseases which require the uptake of such apharmaceutical composition.

As mentioned herein above, not only the herein disclosed conjugates,like drug conjugates or diagnostic conjugates, and/or biologicallyactive, heterologous proteins/protein constructs (comprising theinventive random coil polypeptide or polypeptide segment thereof) are inparticular of medical or pharmaceutical use. Also said random coilpolypeptide or polypeptide segment may be per se employed in such amedical context, for example as “plasma expander” or as blood surrogate,in the amelioration, prevention and/or treatment of a disorder relatedto an impaired blood plasma amount or blood plasma content or in theamelioration, prevention and/or treatment of a disorder related to animpaired blood volume. Disorders that are treated with plasma expandersare, but not limited to, disorders affiliated with blood loss, likeinjuries, surgeries, burns, trauma, or abdominal emergencies,infections, dehydratations etc. Yet, such a medical use is not limitedto the random coil polypeptide or polypeptide segment of this inventionbut can also be extended to certain drug conjugates as disclosed hereinor even to certain biologically active, heterologous proteins/proteinconstructs.

In one embodiment, the composition as described herein may be adiagnostic composition, for example an imaging reagent, optionallyfurther comprising suitable means for detection, wherein said diagnosticcomposition has an increased in vivo and/or in vitro stability.

The compositions of the invention may be in solid or liquid form and maybe, inter alia, in a form of (a) powder(s), (a) tablet(s), (a)solution(s) or (an) aerosol(s). Furthermore, it is envisaged that themedicament of the invention might comprise further biologically activeagents, depending on the intended use of the pharmaceutical composition.

Administration of the suitable (pharmaceutical) compositions may beeffected by different ways, e.g., by parenteral, subcutaneous,intravenous, intraarterial, intraperitoneal, topical, intrabronchial,intrapulmonary and intranasal administration and, if desired for localtreatment, intralesional administration. Parenteral administrationsinclude intraperitoneal, intramuscular, intradermal, subcutaneous,intravenous or intraarterial administration. The compositions of theinvention may also be administered directly to the target site, e.g., bybiolistic delivery to an external or internal target site, like aspecifically effected organ.

Examples of suitable pharmaceutical carriers, excipients and/or diluentsare well known in the art and include phosphate buffered salinesolutions or other buffer solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well knownconventional methods. Suitable carriers may comprise any material which,when combined with the biologically active protein/drug conjugate of theinvention, retains its biological and/or pharmaceutical activity (seeRemington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed,Mack Publishing Company, Easton, Pa.). Preparations for parenteraladministration may include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. The buffers, solvents and/or excipients asemployed in context of the pharmaceutical composition are preferably“physiological” as defined herein above. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesmay include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesmay include fluid and nutrient replenishes, electrolyte replenishers(such as those based on Ringer's dextrose), and the like. Preservativesand other additives may also be present, including antimicrobials,anti-oxidants, chelating agents and/or inert gases and the like. Inaddition, a pharmaceutical composition of the present invention mightcomprise proteinaceous carriers, like, e.g., serum albumin orimmunoglobulin, preferably of human origin.

These pharmaceutical compositions can be administered to the subject ata suitable dose. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depend upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Pharmaceutically activematter may be present in amounts between 1 μg and 20 mg/kg body weightper dose, e.g. between 0.1 mg to 10 mg/kg body weight, e.g. between 0.5mg to 5 mg/kg body weight. If the regimen is a continuous infusion, itshould also be in the range of 1 μg to 10 mg per kilogram of body weightper minute. Yet, doses below or above the indicated exemplary rangesalso are envisioned, especially considering the aforementioned factors.

Furthermore, it is envisaged that the pharmaceutical composition of theinvention might comprise further biologically or pharmaceutically activeagents, depending on the intended use of the pharmaceutical composition.These further biologically or pharmaceutically active agents may be e.g.antibodies, antibody fragments, hormones, growth factors, enzymes,binding molecules, cytokines, chemokines, nucleic acid molecules anddrugs.

It is of note that the present invention is not limited topharmaceutical compositions. Also compositions to be used in research oras diagnostic(s) are envisaged. It is, for example, envisaged that thebiologically active proteins or drug conjugates comprising a random coildomain or component as defined herein, are used in a diagnostic setting.For such a purpose, the inventive biologically active protein or drugconjugate of this invention may be labelled in order to allow detection.Such labels comprise, but are not limited to, radioactive labels (like[³H]hydrogen [¹²⁵I]iodide or [¹²³I]iodide), fluorescent labels(including fluorescent proteins, like green fluorescent protein (GFP) orfluorophores, like fluorescein isothiocyanate (FITC)) or NMR labels(like gadolinium chelates). The here defined labels or markers are in noway limiting and merely represent illustrative examples. The diagnosticcompositions of this invention are particularly useful in tracing orimaging experiments or in a diagnostic medicals setting. In the appendedExamples and Figures, the preparation of a corresponding construct isprovided that comprises conjugates comprising (i) a biosynthetic randomcoil polypeptide or polypeptide segment comprising an amino acidsequence consisting solely of proline and alanine amino acid residues,wherein said amino acid sequence consists of at least 50 proline (Pro)and alanine (Ala) amino acid residues, and (ii) fluorescein ordigoxigenin; see appended FIGS. 13 and 14 and the corresponding figurelegend as well as illustrative Example 22.

But not only pharmaceutical or diagnostic uses of the means and methodsprovided herein are within the gist of the present invention. Thecompounds/conjugates provided herein are also useful in certain otherindustrial areas, like in the food industry, the beverage industry, thecosmetic industry, the oil industry, the paper industry and the like.Therefore, the present invention also provides for uses of thebiosynthetic random coil polypeptide as provided herein in suchindustrial areas. Also part of this invention is, accordingly, a methodfor the production of a cosmetic, of a compound to be used in cosmetictreatments, of a food or of a beverage, said method comprising theculture of the cell comprising a nucleic acid molecule (or a vector)encoding a random coil polypeptide as defined herein or encoding abiologically active protein and/or a biologically active protein and/ora polypeptide that comprises or that is an amino acid sequence that hasor that mediates an activity. Such a method also includes the isolationof said random coil polypeptide, said biologically active protein and/orsaid biologically active protein or said polypeptide that comprises orthat is an amino acid sequence that has or that mediates an activity,like a biological activity, and that additionally comprises said randomcoil polypeptide or random coil polypeptide segment from the culture orfrom said cell. In the same context, other conjugates of interest can beproduced, for example conjugates which are useful in different areas ofindustry, like in the oil or paper industry. The person skilled in theart is readily in a position to adapt the herein provided means andmethods for the generation of corresponding molecular/recombinantconstructs as well as for the generation of conjugates that comprise abiosynthetic random coil polypeptide or polypeptide segment comprisingan amino acid sequence consisting solely of proline and alanine aminoacid residues, wherein said amino acid sequence consists of at least 50proline (Pro) and alanine (Ala) amino acid residues, and a smallmolecule or a polypeptide of interest.

In yet another embodiment, the present invention provides for a kitcomprising the random coil polypeptide (or polypeptide segment thereof),the biologically active protein, the drug conjugate, the nucleic acidmolecule encoding said biologically active protein encoding saidbiologically active protein and/or encoding said biologically activeprotein and/or encoding said polypeptide that comprises or that is anamino acid sequence that has or that mediates an activity (for example abiological activity), the vector comprising said nucleic acid moleculeor the cell comprising said nucleic acid or said vector as describedherein. The kit of the present invention may further comprise, (a)buffer(s), storage solutions and/or additional reagents or materialsrequired for the conduct of medical, scientific or diagnostic assays andpurposes. Furthermore, parts of the kit of the invention can be packagedindividually in vials or bottles or in combination in containers ormulticontainer units.

The kit of the present invention may be advantageously used, inter alia,for carrying out the method of the invention and could be employed in avariety of applications referred herein, e.g., as diagnostic kits, asresearch tools or as medical tools. Additionally, the kit of theinvention may contain means for detection suitable for scientific,medical and/or diagnostic purposes. The manufacture of the kitspreferably follows standard procedures which are known to the personskilled in the art.

The invention is further illustrated by the following, non-limitingFigures and Examples.

FIG. 1. Gene design for the PA#1 Pro/Ala polymer/polypeptide sequence.

Nucleotide and encoded amino acid sequence of a building block for PA#1(SEQ ID NO: 1) obtained by hybridization of two complementaryoligodeoxynucleotides (upper/coding strand oligodeoxynucleotide SEQ IDNO: 17, lower/non-coding strand oligodeoxynucleotide SEQ ID NO: 18). Theresulting nucleic acid has two sticky ends (shown in lower caseletters), corresponding to an Ala codon and anti-codon, respectively,and are mutually compatible. Upon repeated ligation of such a buildingblock, concatamers encoding Pro-Ala polypeptides of varying lengths canbe obtained and subsequently cloned, for example, via (a) SapIrestriction site(s).

FIG. 2. Cloning strategies for a Pro/Ala polymer/polypeptide sequence asfusion to a Fab fragment or to human IFNa2b.

(A) Nucleotide and encoded amino acid sequence stretch (upper/codingstrand SEQ ID NO: 19, lower/non-coding strand SEQ ID NO: 20, encodedamino acid sequence SEQ ID NO: 21) around the C-terminus of theimmunoglobulin light chain of an antibody Fab fragment as encoded onpASK88-Fab-2xSapI (SEQ ID NO: 22), a derivative of pASK75, used forsubcloning of Pro/Ala polymer/polypeptide sequences and expression ofcorresponding biologically active proteins. The nucleotide sequencecarries two SapI recognition sites in mutually reverse orientation,which leads upon digest to protruding DNA ends that are compatible withthe synthetic gene cassette shown in FIG. 1. The recognition sequencesand the C-terminal amino acids of the light chain are underlined.

(B) Nucleotide sequence and encoded amino acid sequence (upper/codingstrand SEQ ID NO: 23, lower/non-coding strand SEQ ID NO: 24, encodedamino acid sequence SEQ ID NO: 25) of a PA#1 polymer/polypeptide with 20residues after insertion of a single cassette as shown in FIG. 1 intothe pASK88-Fab-2xSapI plasmid. Similar ligation/insertion of 10 suchrepeated cassettes resulted in the plasmid vector pFab-PA#1(200) (Seq IDNO: 28) coding for a polymer/polypeptide with 200 residues (SEQ ID NO:26 and 27). The SapI restriction sites flanking the Pro/Alapolymer-encoding sequence are labelled (recognition sequences areunderlined).

(C) Plasmid map of pFab-PA#1(200) (SEQ ID NO: 28). The structural genesfor the heavy chain (HC) and light chain (LC) of the Fab-PA#1(200) areunder transcriptional control of the tetracycline promoter/operator(tet^(p/o)) and the operon ends with the lipoprotein terminator(t_(1pp)). HC comprises the bacterial OmpA signal peptide, the variable(VH) and the first human IgG1 heavy chain constant C domain (CH) as wellas the His₆-tag. LC comprises the bacterial PhoA signal peptide, thevariable (VL) and human light chain constant (CL) domain, the PA#1polymer/polypeptide with 200 residues. The plasmid backbone ofpFab-PA#1(200) outside the expression cassette flanked by the XbaI andHindIII restriction sites is identical with that of the generic cloningand expression vector pASK75 (Skerra (1994) Gene 151:131-135). Singularrestriction sites are indicated.

(D) Nucleotide and amino acid sequence stretch (upper/coding strand SEQID NO: 29, lower/non-coding strand SEQ ID NO: 30, encoded amino acidsequence SEQ ID NO: 31) around the N-terminus of human IFNa2b as clonedon pASK-IFNa2b (SEQ ID NO: 32). The single restriction site SapI thatcan be used for insertion of the Pro/Ala polymer-encoding sequence islabelled (recognition sequence is underlined). The two C-terminal aminoacids of the Strep-tag II are underlined. The first amino acid of themature IFNa2b is labelled with +1.

(E) Nucleotide and encoded amino acid sequence stretch (upper/codingstrand SEQ ID NO: 33, lower/non-coding strand SEQ ID NO: 34, encodedamino acid sequence SEQ ID NO: 35) of the N-terminus of IFNa2b afterinsertion of one PA#1 polymer sequence cassette as shown in FIG. 1. Thesingle restriction site SapI, that remains after insertion of thePro/Ala polymer-encoding sequence, is labelled (recognition sequencesare underlined). The first amino acid of IFNa2b as part of the fusionprotein is labelled (1) and the two C-terminal amino acids of theStrep-tag II are underlined. Similar ligation/insertion of 10 repeatedPA#1 polymer sequence cassettes resulted in the plasmid vectorpPA#1(200)-IFNa2b coding for a polymer/polypeptide with 200 residues(SEQ ID NO: 36)

(F) Plasmid map of pPA#1(200)-IFNa2b (SEQ ID NO: 37). The structuralgene for biologically active protein PA#1(200)-IFNa2b (comprising thebacterial OmpA signal peptide, the Strep-tag II, the PA#1polymer/polypeptide segment with 200 residues, and human IFNa2b) isunder transcriptional control of the tetracycline promoter/operator(tet^(p/o)) and ends with the lipoprotein terminator (t_(1pp)). Theplasmid backbone outside the expression cassette flanked by the XbaI andHindIII restriction sites is identical with that of the generic cloningand expression vector pASK75 (Skerra (1994) loc. cit.). Singularrestriction sites are indicated.

FIG. 3. Analysis of the purified recombinant Fab fragment and thepurified recombinant IFNa2b as well as their Pro/Ala polypeptide/polymerfusions by SDS-PAGE.

The recombinant proteins were produced in E. coli KS272 (Strauch (1988)Proc. Natl. Acad. Sci. USA 85:1576-80) via periplasmic secretion andpurified by means of the His₆-tag (Fab) or the Strep-tag II (IFNa2b)using immobilized metal or streptavidin affinity chromatography,respectively.

(A) Analysis of the purified recombinant Fab and its PA#1 fusion with200 residues by 12% SDS-PAGE. The gel shows 2 μg protein samples each ofFab and Fab-PA#1(200). Samples on the left were reduced with2-mercaptoethanol whereas repeated samples on the right were leftunreduced. Sizes of protein markers—applied under reducingconditions—are indicated on the left margin. Upon reduction of theinterchain disulfide bridge the Fab fragment and its 200 residue PA#1fusion appear as two homogenous bands. In the case of the reduced Fabfragment, the two bands with molecular sizes of ca. 24 and 26 kDa,respectively, correspond to the separated LC and HC. In the case of thereduced Fab-PA#1(200) fusion protein the band at 24 kDa corresponds tothe HC, whereas the band at ca. 90 kDa corresponds to the LC fused withthe PA#1(200) polypeptide segment. Under non-reducing conditions, theFab fragment and its PA#1(200) fusion appear as single homogeneous bandswith apparent molecular sizes of ca. 45 kDa and 100 kDa, respectively.The two apparent size values for the Fab-PA#1(200) fusion protein aresignificantly larger than the calculated masses of 64.3 kDa for thenon-reduced Fab-PA#1(200) and of 39.1 kDa for the isolated LC-PA#1(200).This effect is due to the addition of the Pro/Ala polymer/polypeptidesegment because the Fab fragment itself, with a calculated mass of 48.0kDa, or its unfused light chain exhibit essentially normalelectrophoretic mobility.

(B) Analysis of the purified recombinant IFNa2b and its PA#1 fusionprotein with 200 residues by 12% SDS-PAGE. The gel shows 2 μg proteinsamples each of IFNa2b and of PA#1(200)-IFNa2b. Samples on the left werereduced with 2-mercaptoethanol whereas corresponding samples on theright were left unreduced. Sizes of protein markers—applied underreducing conditions—are indicated on the left margin. The two proteinsappear as single homogeneous bands with apparent molecular sizes of ca.20 kDa and ca. 80 kDa in the reduced form. The latter value issignificantly larger than the calculated mass of 37.0 kDa forPA#1(200)-IFNa2b. This effect is due to the addition of the Pro/Alapolymer/polypeptide segment because the IFNa2b itself, with a calculatedmass of 20.9 kDa, exhibits essentially normal electrophoretic mobility.IFNa2b in the non-reduced state has a slightly higher electrophoreticmobility, indicating a more compact form as a result of its twointramolecular disulfide bridges.

FIG. 4. Quantitative analysis of the hydrodynamic volumes of thepurified recombinant Fab and IFNa2b as well as their PA#1(200) fusions.

(A) Analytical size exclusion chromatography (SEC) of Fab andFab-PA#1(200). 250 μl of the purified protein at a concentration of 0.25mg/ml was applied to a Superdex S200 10/300 GL column equilibrated withPBS buffer. Absorption at 280 nm was monitored and the peak of eachchromatography run was normalized to a value of 1. The arrow indicatesthe void volume of the column (7.8 ml).

(B) Calibration curve for the chromatograms from (A) using a SuperdexS200 10/300 GL column. The logarithm of the molecular weight (MW) ofmarker proteins (cytochrome c, 12.4 kDa; carbonic anhydrase, 29.0 kDa;ovalbumin, 43.0 kDa; bovine serum albumin, 66.3 kDa; alcoholdehydrogenase, 150 kDa, β-amylase, 200 kDa, apo-ferritin, 440 kDa) wasplotted vs. their elution volumes (black circles) and fitted by astraight line. From the observed elution volumes of the Fab fragment andits PA#1 fusion protein (black squares) their apparent molecular sizeswere determined as follows. Fab: 31 kDa (true mass: 48.0 kDa);Fab-PA#1(200): 237 kDa (true mass: 64.3 kDa). These data show thatfusion with the PA#1 polypeptide confers a much enlarged hydrodynamicvolume.

(C) Analytical size exclusion chromatography of IFNa2b andPA#1(200)-IFNa2b. 250 μl of each purified protein at a concentration of0.25 mg/ml was applied to a Superdex S200 10/300 GL column equilibratedwith phosphate-buffered saline, PBS. Absorption at 280 nm was monitoredand the peak of each chromatography run was normalized to a value of 1.The arrow indicates the void volume of the column (7.8 ml).

(D) Calibration curve for the chromatogram from (C) using a SuperdexS200 10/300 GL column. The logarithm of the molecular weight (MW) ofmarker proteins (see B) was plotted vs. their elution volumes (blackcircles) and fitted by a straight line. From the observed elutionvolumes of IFNa2b and its PA#1 fusion protein (black squares) theirapparent molecular sizes were determined as follows. IFNa2b: 22.5 kDa(true mass: 20.9 kDa); PA#1(200)-IFNa2b: 229.0 kDa (true mass: 37.0kDa). These data show that fusion with the PA#1 polypeptide confers amuch enlarged hydrodynamic volume.

FIG. 5. Experimental secondary structure analysis of recombinantproteins and their PA#1 polymer/polypeptide fusions by circulardichroism (CD) spectroscopy.

Spectra were recorded at room temperature in 50 mM K₂SO₄, 20 mMK-phosphate pH 7.5 and normalized to the molar ellipticity, Θ_(M), foreach protein.

(A) CD spectra of the purified recombinant Fab and Fab-PA#1(200). The CDspectrum for the Fab fragment shows the typical features of apredominant β-sheet protein with a broad negative maximum around 216 nm(Sreerama in: Circular Dichroism—Principles and Applications (2000)Berova, Nakanishi and Woody (Eds.) Wiley, New York, N.Y., pp. 601-620),which indicates the correct folding of the bacterially produced Fabfragment. The spectrum of its fusion protein with the Pro/Alapolymer/polypeptide reveals a dominant negative band below 200 nm, whichis indicative of random coil conformation. In addition, there is ashoulder around 220 nm, which results from the β-sheet contribution ofthe Fab fragment and indicates its correct folding even as part of thefusion protein.

(B) Molar difference CD spectrum for Fab-PA#1(200) obtained bysubtraction of the spectrum for the Fab fragment. The difference CDspectrum represents the secondary struture of the 200 residue PA#1polymer/polypeptide segment and reveals a strong minimum around 200 nm,which is a clear indication of random coil conformation in the bufferedaqueous solution (Greenfield (1969) Biochemistry 8: 4108-4116; Sreerama(2000) loc. cit.; Fändrich (2002) EMBO J. 21:5682-5690).

(C) CD spectra of the purified recombinant IFNa2b and PA#1(200)-IFNa2b.The CD spectrum for IFNa2b shows the typical features of a predominantα-helix protein with two negative bands around 208 nm and 220 nm(Sreerama (2000) loc. cit.), which indicates the correct folding of thebacterially produced human IFNa2b. The spectrum of its fusion proteinwith the Pro/Ala polymerr/polypeptide reveals characteristic deviationswith a dominant minimum around 200 nm, which is indicative of randomcoil conformation. In addition, there is a shoulder around 220 nm, whichresults from the α-helical contribution of IFNa2b and indicates thecorrect folding of the IFNa2b even as part of the fusion protein.

(D) Molar difference CD spectrum for PA#1(200)-IFNa2b obtained bysubtraction of the spectrum for IFNa2b. The difference CD spectrumrepresents the secondary struture of the 200 residue PA#1polymer/polypeptide segment and reveals a strong minimum around 200 nm,essentially identical to the one shown in (B). This is again a clearindication of random coil conformation in buffered aqueous solution fora biological polymer comprising Pro and Ala residues according to theinvention.

FIG. 6. Secretory production of a fusion protein between human growthhormone (hGH) and the genetically encoded PA#1 polymer in CHO cells.

(A) Nucleotide and amino acid sequence stretch (upper/coding strand SEQID NO: 38, lower/non-coding strand SEQ ID NO: 39, encoded amino acidsequence SEQ ID NO: 40) around the N-terminus of hGH as cloned onpASK75-His6-hGH (SEQ ID NO: 41). The single restriction sites NheI, thatcan be used together with HindIII (not shown) for subcloning, and SapI,that can be used for insertion of the Pro/Ala polymer-encoding sequence,are labelled (recognition sequence is underlined). The six amino acidsof the His6-tag are underlined. The first amino acid of the hGH islabelled with +1.

(B) Nucleotide and encoded amino acid sequence (upper/coding strand SEQID NO: 42, lower/non-coding strand SEQ ID NO: 43, encoded amino acidsequence SEQ ID NO: 44) of the N-terminus of hGH after insertion of onePA#1 polymer sequence cassette as shown in FIG. 1. The singlerestriction sites NheI, that can be used for subcloning, and SapI, thatremains after insertion of the Pro/Ala polymer-encoding sequence, islabelled (recognition sequences are underlined). The first amino acid ofhGH as part of the fusion protein is labelled (1) and the amino acids ofthe His6-tag are underlined. Similar ligation/insertion of 10 repeatedPA#1 polymer sequence cassettes resulted in the plasmid vectorpASK75-His6-PA#1(200)-hGH coding for the mature fusion protein SEQ IDNO: 45.)

(C) Plasmid map of pASK75-His6-PA#1(200)-hGH (SEQ ID NO: 46). Thestructural gene for biologically active protein His6-PA#1(200)-hGH(comprising the bacterial OmpA signal peptide, the His6-tag, the PA#1polymer/polypeptide segment with 200 residues, and human GH) is undertranscriptional control of the tetracycline promoter/operator(tet^(p/o)) and ends with the lipoprotein terminator (t_(lpp)). Theplasmid backbone outside the expression cassette flanked by the XbaI andHindIII restriction sites is identical with that of the generic cloningand expression vector pASK75 (Skerra (1994) loc. cit.). Singularrestriction sites are indicated.

(D) Plasmid map of pCHO-PA#1(200)-hGH, which encodes aHis6-PA#1(200)-hGH fusion protein (SEQ ID NO: 47). The structural gene,comprising the human growth hormone signal peptide (Sp), the His₆-tag,the PA#1 polymer/polypeptide sequence with 200 residues (PA#1(200)), thehuman growth hormone (hGH), and containing the bovine growth hormonepolyadenylation signal (bGH pA), is under transcriptional control of thecytomegalus virus promoter (CMV^(p)). The singular restriction sitesNheI and HindIII are indicated. The resistance gene forneomycinphosphotransferase (neo) is under control of the SV40 promotor(SV40^(p)) and followed by a SV40 polyadenylation signal (SV40 pA). Inaddition, the plasmid contains the bacterial ColE1 origin of replication(ColE1-ori), the bacteriophage fl origin of replication (fl-ori), andthe β-lactamase gene (bla) to allow propagation and selection in E.coli.

(E) Western blot analysis of a fusion protein between hGH and thegenetically encoded PA#1 polymer of 200 residues produced in CHO cellscompared with recombinant hGH. CHO-K1 cells were transfected either withpCHO-PA#1(200)-hGH (SEQ ID NO: 48) or with pCHO-hGH (SEQ ID NO: 49), asimilar plasmid encoding hGH without the PA#1(200) sequence (but alsocarrying the His6-tag). Two days after transfection, a sample of thecell culture supernatant was subjected to SDS-PAGE and Western blottingwith an anti-hGH antibody conjugated with horse radish peroxidase. Thetwo proteins appear as single bands indicated by arrows, with apparentmolecular sizes of ca. 23 kDa (His6-hGH) and ca. 90 kDa (His6-PA#1-hGH).There is also a weak band around 60 kDa arising from serum proteins inthe culture medium. Whereas the His6-tagged hGH appears at thecalculated mass of 23.5 kDa, the apparent molecular size ofHis6-PA#1-hGH is significantly larger than its calculated mass of 39.5kDa. This effect is due to the hydrophilic random coil nature of thePro-Ala polymer.

FIG. 7. Theoretical prediction of secondary structure for the PA#1Pro/Ala polypeptide/polymer sequence.

This illustration shows the output from the CHOFAS computer algorithmaccording to the Chou-Fasman method (Chou and Fasman (1974) Biochemistry13: 222-245) as implemented on the Sequence Comparison and SecondaryStructure prediction server at the University of Virginia. To avoidboundary effects at the amino and carboxy termini, the 20mer amino acidrepeat according to FIG. 1 was pasted in three consecutive copies(resulting in a concatamer similar as encoded after repeatedligation/insertion of the synthetic gene cassette) and only the outputfor the central 20mer sequence block (boxed) was considered. In the caseof the PA#1 polypeptide sequence/segment (SEQ ID NO: 1) the Chou-Fasmanalgorithm predicts 100% α-helical secondary structure. This is incontrast with the experimentally observed predominant random coilconformation for the PA#1 polypeptide/polypeptide segment as part of afusion protein (see FIG. 5B/D).

FIG. 8: Quantitative analysis of the pharmacokinetics of the purifiedrecombinant Fab fragment and its PA#1 polymer fusions with 200 and 600residues in BALB/c mice.

Plasma samples from Example 16 were assayed for Fab, Fab-PA#1(200), andFab-PA#1(600) concentrations using a sandwich ELISA. To estimate theplasma half-life of Fab, Fab-PA#1(200), and Fab-PA#1(600), the measuredconcentration values were plotted against time post intravenousinjection and numerically fitted assuming a bi-exponential decay. Theunfused Fab fragment exhibited a very fast clearance with an eliminationhalf-life of 1.3±0.1 h. In contrast, the elimination phase determinedfor Fab-PA#1(200) and Fab-PA#1(600) was significantly slower, withterminal half-lives of 4.1±1.8 h and 38.8±11.2 h, respectively, thusdemonstrating a ca. 3-fold and a ca. 30-fold prolonged circulation dueto the Pro/Ala polymer fusion with 200 or 600 residues compared with theunfused Fab fragment.

FIG. 9: Analysis of the purified recombinant Fab fragment as fusion withthe P1A1 or P1A3 polymer having 200 residues.

The recombinant proteins were produced in E. coli KS272 via periplasmicsecretion and purified by means of the His₆-tag using immobilized metalaffinity chromatography. The purified proteins were analyzed by 12%SDS-PAGE. The gel shows 2 μg protein samples each of Fab-P1A1(200) andFab-P1A3(200) as well as, for comparison, of the unfused Fab fragment(cf. FIG. 3A). Samples on the left were reduced with 2-mercaptoethanolwhereas analogous samples on the right were left unreduced. Sizes ofprotein markers—applied under reducing conditions—are indicated on theleft margin. After reduction of the interchain disulfide bridges the Fabfragment and its 200 residue Pro/Ala fusions appear as two homogeneousbands. In the case of the reduced Fab fragment, the two bands withmolecular sizes of ca. 24 and 26 kDa, respectively, correspond to theseparated light chain (LC) and heavy chain fragment (HC). In the case ofthe reduced Fab-P1A1(200) fusion protein the band at 24 kDa correspondsto the HC, whereas the band at ca. 90 kDa corresponds to the LC fusedwith the P1A1(200) polypeptide. In the case of the reduced Fab-P1A3(200)fusion protein the band at 24 kDa corresponds to the HC, whereas theband at ca. 75 kDa corresponds to the LC fused with the P1A5(200)polypeptide. Under non-reducing conditions, the Fab fragment, itsP1A1(200) and its P1A3(200) fusion appear as single prominent bands withapparent molecular sizes of ca. 45 kDa, 110 kDa, and 90 kDa,respectively. The apparent sizes for the Fab-P1A1(200) and Fab-P1A3(200)fusion proteins are significantly larger than the calculated masses of65.3 kDa for the non-reduced Fab-P1A1(200) and of 64.0 kDa for thenon-reduced Fab-P1A3(200). Also, the apparent sizes for thecorresponding reduced light chains are significantly larger than thecalculated masses of 40.7 kDa for the P1A1(200) LC and of 39.4 kDa forthe P1A3(200) LC. This effect is due to the addition of the Pro/Alapolymer/polypeptide segment because the Fab fragment itself, with acalculated mass of 48.0 kDa, or its unfused light chain, with acalculated mass of 23.4 kDa, exhibit essentially normal electrophoreticmobility.

FIG. 10. Quantitative analysis of the hydrodynamic volumes of thepurified recombinant Fab-P1A1(200) and Fab-P1A3(200) fusion proteins.

Analytical size exclusion chromatography (SEC) of Fab-P1A1(200) andFab-P1A3(200). 250 μl of the purified protein at a concentration of 0.25mg/ml was applied to a Superdex S200 10/300 GL column equilibrated withPBS. Absorption at 280 nm was monitored and the peak of eachchromatography run was normalized to a value of 1. The arrow indicatesthe void volume of the column (7.8 ml). From the observed elutionvolumes of the fusion proteins their apparent molecular sizes weredetermined using a similar calibration curve as shown in FIG. 4B asfollows. Fab-P1A1(200): 180.7 kDa (true mass: 65.3 kDa); Fab-P1A3(200):160.2 kDa (true mass: 64.0 kDa). These data show that fusion of aprotein with the P1A1 and/or P1A5 polypeptide confers a much enlargedhydrodynamic volume.

FIG. 11. Experimental secondary structure analysis of Fab-P1A1(200) andFab-P1A3(200) fusions by circular dichroism (CD) spectroscopy.

Spectra were recorded at room temperature in 50 mM K₂SO₄, 20 mMK-phosphate pH 7.5 and normalized to the molar ellipticity, Θ_(M), foreach protein.

(A) CD spectra of the purified recombinant Fab-P1A1(200) andFab-P1A3(200). The CD spectra of the Fab fusion proteins with bothPro/Ala polymers/polypeptides each reveal a dominant negative band below200 nm, which is indicative of random coil conformation. In addition,there is a shoulder around 220 nm, which arises from the β-sheetcontribution of the Fab fragment and indicates its correct folding evenas part of the fusion protein.

(B) Molar difference CD spectra for Fab-P1A1(200) and Fab-P1A3 (200)obtained by subtraction of the spectrum for the unfused Fab fragment(see FIG. 5A). The difference CD spectra represent the secondarystructures of the 200 residue P1A1 (SEQ ID NO: 51) and P1A3 (SEQ ID NO:3) polymers/polypeptide segments, respectively, and reveal a strongminimum around 200 nm, which is a clear indication of their random coilconformation in the buffered aqueous solution (Greenfield (1969)Biochemistry 8: 4108-4116; Sreerama (2000) loc. cit.; Fändrich (2002)EMBO J. 21:5682-5690).

FIG. 12: Preparation of an isolated biosynthetic Pro/Alapolymer/polypeptide.

(A) Plasmid map of pSUMO-PA#1(200) (SEQ ID NO: 60). The structural genefor the fusion protein MK-His(6)-SUMO-PA#1(200) comprising a startmethionine codon followed by a lysine codon, an N-terminal affinity tagof six consecutive His residues, the cleavable small ubiquitin-likemodifier (SUMO) protein Smt3p (Panavas (2009) Methods Mol Biol. 497:303-17), and the PA#1 polymer/polypeptide segment with 200 residues (SEQID NO: 60) is under transcriptional control of the gene 10 promoter ofthe bacteriophage T7 and ends with the tϕ terminator. Additonal plasmidelements comprise the origin of replication (ori), the ampicilinresistance gene (bla), and the fl origin of replication. The plasmidbackbone outside the expression cassette flanked by the NdeI and HindIIIrestriction sites is, except for a SapI restriction site that waseliminated by silent mutation, identical with that of the genericcloning and expression vector pRSET5a (Schoepfer (1993) 124: 83-85).

SEQ ID NO: 60 is provided in the enclosed sequence listing (which isalso part of this description and specification) and is reproducedherein below.

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 60atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 120agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 180ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 240gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 300gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 360tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 420acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 480aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 540cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 600gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 660cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 720tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 780tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 840ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 900tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 960gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1020ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1080tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1140agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1200aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1260cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 1320agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 1380tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 1440gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1500gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 1560ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1620gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 1680ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 1740ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 1800acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 1860gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 1920cggagaagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 1980ggatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc 2040tagaaataat tttgtttaac tttaagaagg agatatacat atgaaacatc accaccatca 2100ccattcggac tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc 2160tgagactcac atcaatttaa aggtgtccga tggatcttca gaaatcttct ttaagatcaa 2220aaagaccact cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat 2280ggactcctta agattcttgt acgacggtat tagaattcaa gctgatcaga cccctgaaga 2340tttggacatg gaggataacg atattattga ggctcacaga gaacagattg gtggcgccgc 2400tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2460tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2520tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2580tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2640tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2700tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2760tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2820tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2880tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgccgc 2940tccagctgca cctgctccag cagcacctgc tgcaccagct ccggctgctc ctgctgcctg 3000aagagcaagc ttgatccggc tgctaacaag cccgaaagga agctgagttg gctgctgcc 3060accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 3120ttgctgaaag gaggaactat atccggatct ggcgtaatag cgaagaggcc cgcaccgatc 3180gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat 3240taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 3300cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 3360aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 3420ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 3480ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 3540caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 3600cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 3660taacgcttac aatttaggtg 3680

(B) Analysis of the bacterially produced His(6)-SUMO-PA#1(200) fusionprotein and its cleavage by 12% SDS-PAGE. The gel shows theSUMO-PAS#1(200) fusion protein extracted from E. coli and purified viaimmobilized metal affinity chromatography (IMAC) and size exclusionchromatography (SEC) before (lane 1) and after proteolytic cleavage withUb1-specific protease 1 (SUMO protease) (lane 2) as described in Example21. All samples were reduced with 2-mercaptoethanol. Sizes of proteinmarkers (M), applied under reducing conditions, are indicated on theleft margin. The His(6)-SUMO-PA#1(200) fusion protein appears as asingle homogeneous band with an apparent molecular size of ca. 100 kDa.Thus, the apparent size for the His(6)-SUMO-PA#1(200) fusion proteinobserved in SDS-PAGE is significantly larger than the calculated mass of28.3 kDa, which is due to the presence of the Pro/Alapolymer/polypeptide. After cleavage, the hydrophilic PA#1(200)polypeptide is not detectably stained by Coomassie blue; hence, only asmall residual fraction of the fusion protein and the cleavedHis(6)-SUMO protein are visible on the SDS polyacrylamide gel (lane 2).The His(6)-SUMO protein shows a homogeneous band with apparent molecularsize of ca. 16 kDa (lane 2) which is well in agreement with itscalculated molecular mass of 12.2 kDa.

FIG. 13: Conjugation of a biosynthetic Pro/Ala polymer/polypeptide withchemical compounds and/or drugs.

(A-D) Production of a fluorescein conjugate with a biosyntheticPA#1(200) polymer/polypeptide (SEQ ID NO: 61) monitored via analyticalsize exclusion chromatography (SEC). The panels show (from top tobottom) SEC runs of purified His(6)-SUMO-PA#1(200) (A),His(6)-SUMO-PA#1(200) after cleavage reaction in the presence of SUMOprotease (B), the cleaved His(6)-SUMO-PA#1(200) batch after chemicalcoupling with a fluorescein NHS ester (C), and the fluorescein-PA#1(200)conjugate after IMAC purification (D). 250 μl of protein/polypeptide ata concentration of ca. 0.5 mg/ml was applied to a Superdex S200 10/300GL column equilibrated with PBS on an Äkta purifier system. Absorptionat 225 nm, 280 nm, and 494 nm was monitored using a UV-900 UV/VISdetector (GE Healthcare) and a prominent peak of each chromatogram wasnormalized to a value of 1. The arrow indicates the void volume of thecolumn (7.3 ml).

(E-K) Characterization of free fluorescein, the biosynthetic PA#1(200)polymer/polypeptide, and its fluorescein conjugate via SEC and UV/VISspectroscopy. The three chromatograms show (from top to bottom) purifiedPA#1(200) (E), the chemical compound fluorescein (F) and the purifiedfluorescein-PA#1(200) conjugate (G). The four UV/VIS spectra show thepurified His(6)-SUMO-PA#1(200) fusion protein (H), the purifiedPA#1(200) polymer/polypeptide (I), free fluorescein (J), and thepurified fluorescein-PA#1(200) conjugate (K) (all in PBS). The arrowsindicate characteric absorption bands/shoulders of SUMO (280 nm),PA#1(200) (225 nm), and fluorescein (494 nm).

(L) Calibration curve for the chromatograms from (A-G) using a SuperdexS200 10/300 GL column. The logarithm of the molecular weight (MW) ofmarker proteins (aprotinin, 6.5 kDa; cytochrome C, 12.4 kDa; carbonicanhydrase, 29.0 kDa; bovine serum albumin, 66.3 kDa; alcoholdehydrogenase, 150 kDa; β-amylase, 200 kDa; apo-ferritin, 440 kDa) wasplotted vs. their elution volumes (x) and fitted by a straight line.From the observed elution volumes of His(6)-SUMO-PA#1(200) (10.81 ml),PA#1(200) (11.51 ml), fluorescein-PA#1(200) (11.49 ml) and fluorescein(27.57 ml), their apparent molecular sizes were determined as follows.His(6)-SUMO-PA#1(200): 215.6 kDa, PA#1(200): 154.1 kDa (true mass: 16.1kDa), fluorescein-PA#1(200): 155.6 kDa (true mass: 16.6 kDa); SUMO: 25.7kDa (true mass: 12.2 kDa); fluorescein: 0.09 kDa (true mass: 0.33 kDa).These data show that fusion with the Pro/Ala polypeptide/polymer confersa much enlarged hydrodynamic volume to the conjugated drug compared withthe unmodified compound.

(M) Characterization of the chemical conjugate between the biosyntheticPA#1(200) polypeptide/polymer and the steroid compound digoxigenin viaElectro Spray Ionisation Mass Spectrometry (ESI-MS). A deconvolutedESI-MS spectrum of digoxigenin-PA#1(200) reveals a mass of 16671.4 Da,which essentially coincides with the calculated mass for thedigoxigenin-PA#1(200) conjugate (16670.6 Da).

FIG. 14: Illustration of chemical conjugates between the biosyntheticPA#1(200) polypeptide/polymer and small molecule drugs.

(A) Fluorescein coupled to the N-terminus of biosynthetic PA#1(200).

(B) Digoxigenin coupled to the N-terminus of biosynthetic PA#1(200).

EXAMPLES

The present invention is additionally described by way of the followingillustrative non-limiting examples that provide a better understandingof the present invention and of its many advantages.

Unless otherwise indicated, established methods of recombinant genetechnology were used as described, for example, in Sambrook (2001) loc.cit.

Example 1: Gene Synthesis for Pro/Ala Amino Acid Polymers/Polypeptides

As described herein above, amino acid repeats consisting of Pro and Alaresidues are depicted herein as Pro/Ala or “PA”. Gene fragments encodinga repetitive polymer sequence comprising Pro and Ala (PA#1 whichcorresponds to SEQ ID NO: 1) were obtained by hybridisation of the twocomplementary oligodeoxynucleotides (SEQ ID NO: 17 and SEQ ID NO: 18)shown in FIG. 1, followed by concatamer formation in a directed mannervia DNA ligation of their mutually compatible but non-palindromic stickyends. Oligodeoxynucleotides were purchased from ThermoScientific (Ulm,Germany) and purified by preparative urea polyacrylamide gelelectrophoresis. The nucleic acid sequences of theoligodesoxynucleotides are depicted in FIG. 1 (SEQ ID NOs 17 and 18comprising an additional GCC codon for alanine, which becomes part ofthe following PA#1 sequence repeat upon ligation of the correspondingsticky ends. Enzymatic phosphorylation was performed by mixing 200 pmolof both oligodeoxynucleotides in 100 μl 50 mM Tris/HCl pH 7.6, 10 mMMgCl₂, 5 mM DTT, 1 mM ATP and incubation for 30 min at 37° C. in thepresence of 10 u polynucleotide kinase (MBI Fermentas, St. Leon-Rot,Germany). After denaturation for 10 min at 80° C., the mixture wascooled to room temperature overnight to achieve hybridization. Then 50μl of this solution was ligated by adding 1 u T4 DNA ligase (MBIFermentas) and 10 μl 100 mM Tris/HCl pH 7.4, 50 mM MgCl₂, 20 mM DTT, 10mM ATP, and in some cases 5 mM of each dATP, dCTP, dGTP, and dTTP, in atotal volume of 100 μl and incubation for 55 min on ice. After 10 minheat inactivation at 70° C. the ligation products were separated by 1.5%(w/v) agarose gel electrophoresis in the presence of TAE buffer (40 mMTris, 20 mM acetic acid, 1 mM EDTA). After staining with ethidiumbromide the band corresponding to the assembled gene segment of 300 bplength was excised and isolated.

Example 2: Construction of PFab-PA#1(200) as Expression Vector for aFab-PA#1 Fusion Protein

For cloning of a 10mer repeat of the synthetic gene fragment coding forthe 20 amino acid sequence of PA#1 from Example 1 the plasmid vectorpASK88-Fab-2xSapI (SEQ ID NO: 22), an expression plasmid for an Fabfragment (Schlapschy (2007) Protein Eng. Des. Sel. 20:273-284) harboringa nucleotide sequence with two SapI restriction sites in reversecomplementary orientation at the 3-′end of the light chain (FIG. 2A),was employed. This vector, which is a derivative of pASK75 (Skerra, A.(1994) Gene 151:131-135), was cut with SapI, dephosphorylated withshrimp alkaline phosphatase (USB, Cleveland, Ohio), and ligated with a300 bp cassette of the synthetic DNA fragment obtained from Example 1.The resulting intermediate plasmid pFab-PA#1(100) was again cut withSapI, dephosphorylated with shrimp alkaline phosphatase, and ligatedwith a 300 bp cassette of the synthetic DNA fragment obtained fromExample 1 (as exemplified in FIG. 2B, however with only a PA#1(20)polymer/polypeptide cassette). The resulting plasmid was designatedpFab-PA#1(200) (SEQ ID NO: 28) (FIG. 2C). It should be noted that onthis plasmid the coding region for the 200 residue PA#1 sequence repeatwas flanked by two SapI restriction, which enables precise excision andfurther subcloning of the entire sequence cassette, carrying 5′-GCCnucleotide overhangs.

After transformation of E. coli XL1-Blue (Bullock (1987) Biotechniques5: 376-378), plasmid was prepared and the sequence of the clonedsynthetic nucleic acid insert was confirmed by restriction analysis anddouble-stranded DNA sequencing (ABI-Prism™310 Genetic analyzer,Perkin-Elmer Applied Biosystems, Weiterstadt, Germany) using the BigDye™terminator kit as well as oligodeoxynucleotide primers that enabledsequencing from both sides.

Example 3: Construction of PASK-PA#1(200)-IFNa2b as an Expression Vectorfor a PA#1(200)-IFNa2b Fusion Protein

For the construction of an expression plasmid encoding IFNa2b as fusionwith a 200 residue PA#1 sequence repeat, PA#1(200), pASK-IFNa2b (SEQ IDNO: 32) (FIG. 2D) was cut with SapI, dephosphorylated with shrimpalkaline phosphatase, and ligated with the gene fragment encoding the200 residue PA#1 polypeptide excised from the previously constructedplasmid pFab-PA#1(200) (Example 2) by restriction digest with SapI (asexemplified in FIG. 2E, however with only a PA#1(20) polymer/polypeptidecassette). After transformation of E. coli JM83 (Yanisch-Perron. (1985)Gene 33:103-119), plasmid was prepared and the presence of the correctinsert was confirmed by restriction analysis. The resulting plasmid wasdesignated pPA#1(200)-IFNa2b (SEQ ID NO: 37) (FIG. 2F).

Example 4: Bacterial Production and Purification of Fusion ProteinsBetween an Fab Fragment and a Genetically Encoded PA#1Polymer/Polypeptide

The Fab fragment (calculated mass: 48.0 kDa) and the Fab-PA#1(200)fusion (calculated mass: 64.3 kDa) were produced at 22° C. in E. coliKS272 harboring the corresponding expression plasmids from Example 3,together with the folding helper plasmid pTUM4 (Schlapschy (2006)Protein Eng. Des. Sel. 19:385-390), using shaker flask cultures with 2 LLB medium containing 100 mg/l ampicillin and 30 mg/l chloramphenicol.Induction of recombinant gene expression was performed by addition of0.4 mg anhydrotetracycline at OD₅₅₀=0.5 over night (typically resultingin OD₅₅₀ of ca. 1.0 at harvest). Periplasmic extraction in the presenceof 500 mM sucrose, 1 mM EDTA, 100 mM Tris/HCl pH 8.0 containing 50 μg/mllysozyme was performed as described elsewhere (Breustedt (2005) Biochim.Biophys. Acta 1764:161-173) and followed by purification by means of theHis₆-tag using immobilized metal affinity chromatography (Skerra (1994)Gene 141: 79-84) with an imidazole gradient from 0 to 200 mM in 500 mMbetaine, 50 mM Na-phosphate pH 7.5).

Homogeneous protein preparations were obtained for both recombinant Fabfragments (FIG. 3A) with yields of 0.2 mg L⁻¹ OD⁻¹ for the unfused Faband 0.1 mg L⁻¹ OD⁻¹ for Fab-PA#1(200). SDS-PAGE was performed using ahigh molarity Tris buffer system (Fling (1986) Anal. Biochem. 155:83-88). Protein concentrations were determined according to theabsorption at 280 nm using calculated extinction coefficients (Gill(1989) Anal. Biochem. 182: 319-326) of 68290 M⁻¹ cm⁻¹ both for theunfused Fab and its PA#1 polymer fusion as the Pro/Ala polymer did notcontribute to UV absorption because of its lack of aromatic amino acids.

Example 5: Bacterial Production and Purification of Fusion ProteinsBetween IFNa2b and a Genetically Encoded PA#1 Polymer/Polypeptide

IFNa2b (calculated mass: 20.9 kDa) and PA#1(200)-IFNa2b (calculatedmass: 37.0 kDa) were produced at 22° C. in E. coli KS272 harboring thecorresponding expression plasmids from Example 3, together with thefolding helper plasmid pTUM4 (Schlapschy (2006) loc. cit.), using shakerflask cultures with 2 L LB medium containing 100 mg/l ampicillin and 30mg/l chloramphenicol. Induction of recombinant gene expression wasperformed by addition of 0.4 mg anhydrotetracycline at OD₅₅₀=0.5 overnight (typically resulting in OD₅₅₀ of ca. 1.0 at harvest). Periplasmicextraction in the presence of 500 mM sucrose, 1 mM EDTA, 100 mM Tris/HClpH 8.0 containing 50 μg/ml lysozyme was performed as described elsewhere(Breustedt (2005) loc. cit.) and followed by purification via theStrep-tag II using streptavidin affinity chromatography (Schmidt (2007)Nat. Protoc. 2:1528-1535) in the presence of 150 mM NaCl mM EDTA, 100 mMTris/HCl pH 8.0.

Homogeneous protein preparations were obtained for both recombinantIFNa2b proteins (FIG. 3B) with yields of 0.15 mg L⁻¹ OD⁻¹ for IFNa2b and0.1 mg L⁻¹ OD⁻¹ for PA#1(200)-IFNa2b. SDS-PAGE was performed using ahigh molarity Tris buffer system (Fling (1986) loc. cit.). Proteinconcentrations were determined according to the absorption at 280 nmusing calculated extinction coefficients (Gill (1989) loc. cit.) of23590 M⁻¹ cm⁻¹ both for the unfused IFNa2b and its PA#1 polymer fusion.

Example 6: Measurement of the Hydrodynamic Volume for the RecombinantFusion Protein Between a Fab Fragment and a Genetically Encoded PA#1Polymer of 200 Residues By Analytical Gel Filtration

Size exclusion chromatography (SEC) was carried out on a Superdex S200HR 10/300 GL column (GE Healthcare Europe, Freiburg, Germany) at a flowrate of 1 ml/min using an Äkta Purifier 10 system (GE Healthcare) withPBS (115 mM NaCl, 4 mM KH₂PO₄, 16 mM Na₂HPO₄; pH 7.4) as running buffer.250 μl samples of the purified Fab fragment and its 200 residue PA#1fusion, obtained from the metal affinity affinity chromatography asdescribed in Example 4, were individually applied at a concentration of0.25 mg/ml in PBS. Both proteins eluted in a single homogenous peak asshown in FIG. 4A.

For column calibration (FIG. 4B), 250 μl of an appropriate mixture ofthe following globular proteins (Sigma, Deisenhofen, Germany) wereapplied in PBS at protein concentrations between 0.2 mg/ml and 0.5mg/ml: cytochrome c, 12.4 kDa; carbonic anhydrase, 29.0 kDa; ovalbumin,43.0 kDa; bovine serum albumin, 66.3 kDa; alcohol dehydrogenase, 150kDa; β-amylase, 200 kDa; apo-ferritin, 440 kDa.

As result, the fusion protein with the 200 residue PA#1polymer/polypeptide exhibited a significantly larger size thancorresponding globular proteins with the same molecular weight. Theapparent size increase for Fab-PA#1(200) was 7.4-fold compared with theunfused Fab fragment whereas the true mass was only larger by 1.3-fold.This observation clearly indicates a much increased hydrodynamic volumeconferred to the biologically active Fab fragment by the Pro/Alapolypeptide segment according to this invention.

Example 7: Measurement of the Hydrodynamic Volume for the RecombinantFusion Protein Between IFNa2b and a Genetically Encoded PA#1 Polymer of200 Residues by Analytical Gel Filtration

Size exclusion chromatography was carried out with IFNa2b andPA#1(200)-IFNa2b on a Superdex S200 HR 10/300 GL column (GE Healthcare)at a flow rate of 1 ml/min using an Äkta Purifier 10 system (GEHealthcare) similarly as described in Example 6. Both proteins eluted ina single homogenous peak as shown in FIG. 4C.

As result, the fusion protein with the 200 residue PA#1polymer/polypeptide exhibited a significantly larger size thancorresponding globular proteins with the same molecular weight (FIG.4D). The apparent size increase for PA#1(200)-IFNa2b was 10.2-foldcompared with the unfused IFNa2b protein whereas the true mass was onlylarger by 1.8-fold. This observation clearly indicates a much increasedhydrodynamic volume conferred to the biologically active interferon bythe Pro/Ala polymer/polypeptide according to this invention.

Example 8: Detection of Random Coil Conformation for the BiosyntheticPA#1 Polymer Fused to a Fab Fragment Via Circular Dichroism Spectroscopy

Secondary structure was analysed using a J-810 spectropolarimeter(Jasco, Groβ-Umstadt, Germany) equipped with a quartz cuvette 106-QS(0.1 mm path length; Hellma, Müllheim, Germany). Spectra were recordedfrom 190 to 250 nm at room temperature by accumulating 16 runs(bandwidth 1 nm, scan speed 100 nm/min, response 4 s) using 3.12 to 15.4μM protein solutions obtained from Example 4 in 50 mM K₂SO₄, 20 mMK-phosphate pH 7.5. After correction for solution blanks, spectra weresmoothed using the instrument software, and the molar ellipticity Θ_(M)was calculated according to the equation:

$\Theta_{M} = \frac{\Theta_{obs}}{c \cdot d}$whereby Θ_(obs) denotes the measured ellipticity, c the proteinconcentration [mol/l], d the path length of the quartz cuvette [cm]. TheΘ_(M) values were plotted against the wavelength using Kaleidagraph(Synergy Software, Reading, Pa.).

The measured circular dichroism (CD) spectrum for the recombinant Fabwas in accordance with the β-sheet dominated immunglobuline fold,whereas the spectrum for the Fab-PA#1(200) fusion protein revealed asignificant contribution of random coil conformation (FIG. 5A). Toanalyze the spectroscopic contribution by the Pro/Ala polypeptidesegment in greater detail the molar difference CD spectrum with respectto the unfused Fab fragment was calculated (FIG. 5B) by subtraction ofthe latter spectrum from the one for Fab-PA#1(200). As result, a strongminimum around 200 nm, which is characteristic of random coilconformation, was observed. Thus, the Pro/Ala sequence as part of therecombinant fusion protein appears to be present as a random coilpolymer under physiological buffer conditions.

Example 9: Detection of Random Coil Conformation for the GeneticallyEncoded PA#1 Polymer Fused to IFNa2b Via Circular Dichroism Spectroscopy

Secondary structure was analysed by CD measurements for IFNa2b andPA#1(200)-IFNa2b (obtained from Example 5) as described in Example 8using 3.6 to 38.7 μM protein solutions. The spectrum of PA#1(200)-IFNa2brevealed significant contributions of α-helical secondary structure,indicative of the known α-helix bundle fold of interferon, as well as ofrandom coil conformation (FIG. 5C). To analyze the spectroscopiccontributions by the Pro/Ala polymer fusion partner in greater detailthe molar difference CD spectrum with respect to the unfused IFNa2b wascalculated by subtraction of the two individual spectra (FIG. 5D). Asresult, a strong minimum around 200 nm characteristic of random coilconformation was observed. Thus, the Pro/Ala polypeptide segment as partof the recombinant fusion protein appears to be present as a random coilpolymer under aqueous buffer conditions.

Example 10: Quantitative Analysis of the Secondary Structure of the FabFragment, of IFNa2b and of Their 200 Residue PA#1 Polymer Fusions

The secondary structure content of the Fab fragment, Fab-PA#1(200),IFNa2b, and PA#1(200)-IFNa2b was individually quantified from thecorresponding CD spectra measured in Examples 8 and 9 using thesecondary structure deconvolution program CDNN ver. 2.1 (Böhm (1992)Protein Eng. 5:191-195) with a set of 33 base spectra for thedeconvolution of complex CD spectra The results of this analysis areprovided in the following Table:

PA#1 Fab- Diff: (100)- Diff: Fab PA#1(200) PA#1(200) IFNa2b IFNa2bPA#1(200) α-helix 9.5% 7.5%  2.1% 38.2% 31.0% 0.7% anti-parallel 40.4%3.1%    0% 1.8% 0.2% 4.6% β-sheet parallel 6.9% 1.3%  0.3% 8.4% 0.7%0.6% β-sheet β-turn 6.2% 50.4%  78.6% 19.2% 75.2% 69.7% random coil37.2% 63.4%  94.8% 35.9% 64.4% 97.5% Σ total 100.2% 125.8% 175.8% 103.5%171.4% 170.0% Σ β-turn and 43.4% 113.8% 173.4% 55.1% 139.6% 169.1%random coil

Compared with the predominantly β-sheet secondary structure content ofthe recombinant Fab fragment, which is in accordance with its knownimmunoglobulin fold (see Eigenbrot (1993) J. Mol. Biol. 229:969-995),the fraction of unstructured conformation (comprising random coil andβ-turn) clearly increases if the PA#1 polymer is fused to the Fabfragment. The difference CD spectrum for the Pro/Ala polypeptide segmentreveals a clear random coil conformation. Analysis of the secondarystructure shows the presence of a high fraction of unstructuredconformations (comprising random coil and β-turn) which nearly comprise100% of the total secondary structure. Similarly, compared with thepredominantly α-helical secondary structure content of the recombinantIFNa2b, which is in accordance with its known three-dimensionalstructure as an α-helix bundle protein (Radhakrishnan (1996) Structure4:1453-1463), the fraction of unstructured conformation for the wholeprotein clearly increases if the PA#1 polymer is fused to IFNa2b. Thedifference CD spectrum for the Pro/Ala polypeptide segment reveals aclear random coil conformation. Analysis of the secondary structureshows the presence of a high fraction of unstructured conformations(comprising random coil and (β-turn) which nearly comprise 100% of thetotal secondary structure.

Different results were obtained when a theoretical analysis of the PA#1polymer sequence was performed using the Chou-Fasman algorithm (Chou andFasman (1974) Biochemistry 13: 222-245). The results of this analysisare illustrated in FIG. 7. This algorithm predicts 100% α-helicalsecondary structure, which is in clear contrast with the experimentaldata. Thus, this algorithm is not useful to confidently predictunstructured conformation for an amino acid polymer according to theinvention.

Example 11: Construction of PASK75-His6-PA#1(200)-hGH as an ExpressionVector for a His6-PA#1(200)-hGH Fusion Protein

For the construction of an expression plasmid encoding hGH as fusionwith a 200 residue PA#1 sequence repeat, PA#1(200), pASK75-His6-hGH (SEQID NO: 41) (FIG. 6A) was cut with SapI, dephosphorylated with shrimpalkaline phosphatase, and ligated with the gene fragment encoding the200 residue PA#1 polypeptide excised from the previously constructedplasmid pFab-PA#1(200) (Example 2) by restriction digest with SapI (asexemplified in FIG. 6B, with only a PA#1(20) polymer/polypeptidecassette). After transformation of E. coli JM83 (Yanisch-Perron. (1985)loc. cit.), plasmid was prepared and the presence of the correct insertwas confirmed by restriction analysis. The resulting plasmid wasdesignated pASK75-His6-PA#1(200)-hGH (SEQ ID NO: 46) (FIG. 6C).

Example 12: Construction of an Expression Vector for the SecretoryProduction of Human Growth Hormone Fused with a 200 Residue PA#1Polymer/Polypeptide in Chinese Hamster Ovary Cells

The vector pASK75-His6-PA#1(200)-hGH (SEQ ID NO: 46), a derivative ofpASK75 (Skerra (1994) loc. cit.), allowing prokaryotic production of thehGH PA#1 fusion protein, was cut with NheI and HindIII. This fragmentwas purified via agarose gel electrophoresis and ligated with thecorrespondingly cut vector pCHO (SEQ ID NO: 50). After transformation ofE. coli XL1-Blue (Bullock (1987) loc. cit.), plasmid was prepared andthe correct insertion of the fragment was verified via restrictionanalysis. The resulting plasmid, which codes for the hGH signal peptidefused to the His₆ tag, a PA#1(200) polypeptide segment, and the humangrowth hormone (hGH), was designated pCHO-PA#1(200)-hGH SEQ ID NO: 48)and is depicted in FIG. 6D.

Example 13: Secretory Production of a Fusion Protein Between HumanGrowth Hormone (hGH) and the Genetically Encoded PA#1 Polymer in CHOCells

CHO-K1 cells ATCC No. CCL-61 were cultured in Quantum 263 medium (PAALaboratories, Colbe, Germany) in a 100 mm plastic dish until 50%confluency was reached. Cells were transfected with 8 μgpCHO-PA#1(200)-hGH (SEQ ID NO: 48) or, for control, pCHO-hGH (SEQ ID NO:49), a similar plasmid encoding hGH without the PA#1(200) sequence,using the Nanofectin Kit (PAA Laboratories, Colbe, Germany). After 6 h,cell culture medium was exchanged by 7 ml Opti-MEM®-I reduced serummedium (Invitrogen, Darmstadt, Germany) and cells were incubated at 37°C. in a humidified atmosphere with 5% CO₂. After two days, 20 μl of thecell culture supernatant was taken and diluted with 5 μl SDS-PAGEloading buffer containing β-mercaptoethanol. After 5 min heating at 95°C., 15 μl of each sample was subjected to 12% SDS-PAGE. Followingelectro-transfer onto a nitrocellulose membrane (Schleicher & Schuell,Dassel, Germany) by means of a semi-dry blotting apparatus, the membranewas washed 3 times for 15 min with 10 ml PBST (PBS containing 0.1% v/vTween 20). The membrane was incubated with 10 ml of a 1:1000 dilution ofanti human growth hormone antibody ab1956 conjugated with horse radishperoxidase (Abcam, Cambridge, UK). After incubation for 1 h and washingthe membrane twice for 5 min with 20 ml PBST and twice for 5 min withPBS, the chromogenic reaction was performed in the presence of 15 ml ofSIGMAFAST™ 3,3-diaminobenzidine solution (Sigma-Aldrich Chemie, Munich,Germany). The reaction was stopped by washing with water and air-dryingof the membrane. The blot revealed signals for both recombinant proteinsamples (FIG. 6E), thus proving secretory production of the hGH fusionprotein with the PA#1 polypeptide in CHO cells.

Example 14: Bacterial Production and Purification of Fusion ProteinsBetween HGH and a Genetically Encoded PA#1 Polymer/Polypeptide

Human growth hormone (hGH) (calculated mass: 23.4 kDa), PA#1(200)-hGH(calculated mass: 39.6 kDa), PA#1(400)-hGH (calculated mass: 55.8 kDa)and PA#1(600)-hGH (calculated mass: 72.0 kDa) were produced in E. coliKS272 harboring the corresponding expression plasmids from Example 11 ortheir derivatives with a double (encoding 400 residues) or triple (600residues) PA#1 sequence cassette, respectively. Bacterial production wasperformed at 22° C. in shaker flask cultures with 2 L LB mediumcontaining 2.5 g/L glucose, 0.5 g/L proline and 100 mg/l ampicillin.Induction of recombinant gene expression was performed by addition of0.4 mg anhydrotetracycline at OD₅₅₀=0.5 for 3 h. Periplasmic extractionin the presence of 500 mM sucrose, 1 mM EDTA, 100 mM Tris/HCl pH 8.0containing 50 μg/ml lysozyme was carried out as described elsewhere(Breustedt (2005) loc. cit.) and followed by purification via theHis₆-tag using the HisTrap High Performance affinity column (GEHealthcare) with 40 mM Na-phosphate pH 7.5, 0.5 M NaCl as buffer.Proteins were eluted using an imidazole concentration gradient from 0 to150 mM (dissolved in the running buffer and adjusted with HCl to pH 7.5)and further purified via size exclusion chromatography using a Superdex200-HR10/30 column (GE Healthcare) equilibrated with PBS (115 mM NaCl, 4mM KH₂PO₄, 16 mM Na₂HPO₄, pH 7.4).

After size exclusion chromatography homogeneous protein preparationswere obtained for all recombinant hGH fusion proteins without signs ofaggregation and with yields of 1 mg L⁻¹ OD⁻¹ for hGH, 0.3 mg L⁻¹ OD⁻¹for PA#1(200)-hGH, 0.3 mg L⁻¹ OD⁻¹ for PA#1(400)-hGH and 0.2 mg L⁻¹ OD⁻¹for PA#1(600)-hGH. SDS-PAGE was performed using a high molarity Trisbuffer system (Fling (1986) loc. cit.). Protein concentrations weredetermined according to the absorption at 280 nm using calculatedextinction coefficients (Gill (1989) loc. cit.) of 16050 M⁻¹ cm⁻¹ forthe unfused hGH and all its PA#1 polypeptide fusions.

Example 15: Measurement of Binding Affinity of Human Growth Hormone andits PA#1 Polymer Fusions Towards the Extracellular Domain of HumanGrowth Hormone Receptor Using Surface Plasmon Resonance

The affinity of hGH and its PA#1 polypeptide fusions to a human growthhormone receptor Fc fusion protein (hGHR-Fc; R&D Systems) was determinedvia surface plasmon resonance (SPR) real time measurements on a Biacore2000 system (GE Healthcare). First, 15 μl mouse anti-human IgG-Fccapture antibody (Jackson Immuno Research) at a concentration of 100μg/m in 10 mM Na-acetate pH 5.0 was immobilized to the surface of twoflow channels of a CMDP chip (XanTec bioanalytics) using an aminecoupling kit (GE Healthcare). This resulted in ca. 2700 response units(RU). After equilibration with PBS/T (PBS containing 0.05% (v/v) Tween20) as flow buffer, one channel of the chip was charged with 2 μg/mlhGHR-Fc at a flow rate of 5 μl/min until an additional signal of ca. 300RU was reached. Then, 75 μl of hGH or its PA#1 polypeptide fusions inPBS/T was injected at varying concentrations and the association anddissociation phases were measured under continuous buffer flow of 20μl/min. For regeneration, three 6 μl pulses of 10 mM glycine/HCl pH 2.7were applied. The sensograms were corrected by double subtraction of thecorresponding signals measured for the channel without immobilizedreceptor and an averaged baseline determined from several buffer blankinjections (Myszka (1999) Mol. Recognit. 12: 279-284). Kinetic dataevaluation was performed by a global fit of the traces from at leastseven different sample injections according to the 1:1 Langmuir bindingmodel using BlAevaluation software version 3.1 (GE Healthcare). Thevalues obtained from SPR measurements for the kinetic and derivedequilibrium constants of the complexes between hGH or its PA#1 fusionsand the human growth hormone receptor are summarized in the followingTable:

hGH variant k_(on) [10⁵ M⁻¹ s⁻¹] k_(off) [10⁻⁶ s⁻¹] K_(D) [pM] hGH 10.210.6 10.4 PA#1(200)-hGH 4.75 9.18 19.3 PA#1(400)-hGH 3.26 14.0 42.9PA#1(600)-hGH 3.29 12.5 38.0

These data show that the fusion of hGH with PA#1 polypeptides ofdifferent lengths does not significantly interfere with receptorbinding. All hGH PA#1 polypeptide fusions retain receptor bindingactivity within a factor 5 compared with the recombinant hGH lacking aPA#1 polypeptide.

Example 16: Detection of Prolonged Plasma Half-Life In Vivo for theRecombinant Fusion Proteins Between a Fab Fragment and GeneticallyEncoded PA#1 Polymers

Adult BALB/c mice (SPF stock breeding; TU München, Freising, Germany)were intravenously injected according to the following Table:

Group A B D Test item Fab Fab-PA#1(200) Fab-PA#1(600) Administrationroute intravenous Dose [mg/kg b.w.] 5.0 5.0 5.0 Concentration [mg/ml]1.0 1.0 1.0 Application volume [ml/kg b.w.] 5.0 No. of animals/group 9 99 No. of blood sampling time 12 12 12 points No. of animals/samplingtime 3 3 3 point No. of blood samplings/animal 4/1 4/1 4/1

The total volume of intravenously administered test item was calculatedaccording to the individual body weight (b.w.) recorded on the day ofadministration (e.g. an animal with 20 g body weight received 100 μl of1 mg/ml test item). Blood sampling was performed according to thefollowing Table:

Time points for blood sampling after injection Test item Subgroup 10 min30 min 1 h 2 h 3 h 4 h 6 h 8 h 12 h 24 h 36 h 48 h Fab 1 x x x xFab-PA#1(200) x x x x Fab-PA#1(600) x x x x 2 x x x x x x x x x x x x 3x x x x x x x x x x x x

For each substance (Test item) altogether nine animals—divided intothree subgroups 1-3 with each three animals—were injected, eachproviding four samples at different time points. Blood samples(approximately 50 μl) were taken from the tail vene and stored at 4° C.for 30 min. After centrifugation for 10 min at 10 000 g and 4° C. thesupernatant (plasma) was immediately frozen and stored at −20° C.

For quantitative detection of the Fab fusion protein in an ELISA, thewells of a 96 well microtiter plate (Maxisorb, NUNC, Denmark) werecoated overnight at 4° C. with 50 μl of a 10 μg/ml solution ofrecombinant Her2/ErbB2 ectodomain antigen in 50 mM NaHCO₃ pH 9.6. Then,the wells were blocked with 200 μl of 3% (w/v) BSA in PBS for 1 h andwashed three times with PBS/T (PBS containing 0.1% (v/v) Tween 20). Theplasma samples were applied in dilution series in PBS/T containing 0.5%(v/v) mouse plasma from an untreated animal and incubated for 1 h. Thewells were then washed three times with PBS/T and incubated for 1 h with50 μl of a 1:1000 diluted solution of an anti-human CK antibody alkalinephosphatase conjugate in PBS/T. After washing twice with PBS/T and twicewith PBS the chromogenic reaction was started by adding 50 μl of 0.5μg/ml p-nitrophenyl phosphate in 100 mM Tris/HCl pH 8.8, 100 mM NaCl, 5mM MgCl₂ as substrate, and after 15 min at 25° C. the absorbance at 405nm was measured. Concentrations of Fab, Fab-PA#1(200), and Fab-PA#1(600)in the plasma samples were quantified by comparison of the measuredsignals with standard curves which were determined for dilution seriesfor the corresponding purified proteins at defined concentrations inPBS/T containing 0.5% (v/v) mouse plasma from untreated animals.

To estimate the plasma half-life of Fab, Fab-PA#1(200), andFab-PA#1(600), the concentration values, c(t), were determined for eachtime point from the ELISA measurements and plotted against time postintravenous injection, t. These data were numerically fitted usingKaleidaGraph software assuming a bi-exponential decay according to theequation

$\quad\mspace{11mu}{\quad\mspace{11mu}{{c(t)} = {{c_{\alpha}e^{{- \ln}\; 2\frac{t}{\tau_{1/2}^{\alpha}}}} + {\left( {c_{0} - c_{\alpha}} \right)e^{{- \ln}\; 2\frac{t}{\tau_{1/2}^{\beta}}}}}}}$whereby τ^(α) _(1/2) and τ^(β) _(1/2) are the half-life values of thedistribution phase α and the elimination phase β, respectively. c₀ isthe total blood concentration at time point zero while c_(α) is theconcentration amplitude for the distribution phase.

FIG. 8 depicts the pharmacokinetics for the three test items in BALB/cmice. While the recombinant Fab shows a rapid blood clearance with anelimination half-life of just ca. 1.3 h, the Fab-PA#1(200) andFab-PA#1(600) fusion proteins have a more than 3-fold and 29-foldextended half-life with corresponding values of ca. 4.1 h and 38.8 h,respectively. These data prove that the in vivo plasma half-life of aFab fragment is significantly prolonged due to fusion with a Pro/Alapolymer/polypeptide, whereby the half-life becomes longer withincreasing length of the amino acid polymer.

Example 17: Gene Synthesis for P1A1 and P1A3 Amino AcidPolymers/Polypeptides and Construction of PFab-P1A1(200) andPFab-P1A3(200) as Expression Vectors for Fab-P1A1(200) and Fab-P1A3(200)Fusion Proteins

Gene fragments encoding a repetitive polymer sequence comprising thePro/Ala polypeptides/polymers P1A1 (SEQ ID NO: 51) and P1A3, alsodesignated PA#3, (SEQ ID NO: 3) were obtained by hybridisation of pairsof complementary oligodeoxynucleotides, respectively, SEQ ID NO: 52 andSEQ ID NO: 53 for P1A1 and SEQ ID NO: 54 and SEQ ID NO: 55 for P1A3 asdescribed in Example 1. pFab-P1A1(200) (Seq ID NO: 58) andpFab-P1A3(200) (Seq ID NO: 59) coding for Fab fragments with thecorresponding Pro/Ala polymers/polypeptide segments of 200 residues atthe C-terminus of the light chain (LC) (amino acid sequence of LCFab-P1A1(200): SEQ ID NO: 56; amino acid sequence of LC Fab-P1A3(200):SEQ ID NO: 57) were constructed in an analogous manner topFab-PA#1(200), which has been described in Example 2.

In the following SEQ ID NOs: 56, 57, 58 and 59 are also reproduced.However, these sequences are also comprised in the appended sequencelisting which is a specific part of this disclosure and the descriptionof the present invention.

SEQ ID NO: 56Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1               5                   10                  15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala            20                  25                  30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile        35                  40                  45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly    50                  55                  60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65                  70                  75                  80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro                85                  90                  95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala            100                 105                 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly        115                 120                 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala    130                 135                 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145                 150                 155                 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                165                 170                 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr            180                 185                 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser        195                 200                 205Phe Asn Arg Gly Glu Cys Ser Ser Ala Pro Ala Pro Ala Pro Ala Pro    210                 215                 220Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro225                 230                 235                 240Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro                245                 250                 255Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro            260                 265                 270Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro        275                 280                 285Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro    290                 295                 300Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro305                 310                 315                 320Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro                325                 330                 335 Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro            340                 345                 350Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro        355                 360                 365Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro    370                 375                 380Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro385                 390                 395                 400Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro                405                 410                 415 AlaSEQ ID NO: 57Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1               5                   10                  15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala            20                  25                  30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile        35                  40                  45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly    50                  55                  60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65                  70                  75                  80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro                85                  90                  95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala            100                 105                 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly        115                 120                 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala    130                 135                 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145                 150                 155                 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                165                 170                 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr            180                 185                 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser        195                 200                 205Phe Asn Arg Gly Glu Cys Ser Ser Ala Ala Ala Pro Ala Ala Ala Pro    210                 215                 220Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro225                 230                 235                 240Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro                245                 250                 255Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro            260                 265                 270Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro        275                 280                 285Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro    290                 295                 300Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro305                 310                 315                 320Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro                325                 330                 335Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro            340                 345                 350Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro        355                 360                 365Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro    370                 375                 380Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro385                 390                 395                 400Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala Ala Pro                405                 410                 415 AlaSEQ ID NO: 58acccgacacc atcgaatggc cagatgatta attcctaatt tttgttgaca ctctatcatt   60gatagagtta ttttaccact ccctatcagt gatagagaaa agtgaaatga atagttcgac  120aaaaatctag ataacgaggg caaaaaatga aaaagacagc tatcgcgatt gcagtggcac  180tggctggttt cgctaccgta gcgcaggccg aagttaaact gcaggaatcc ggtggtggtc  240tggttcagcc aggtggttcc ctgcggctct cgtgtgctgc ttccggtttc aacatcaaag  300acacctacat ccactgggtt cgtcaggctc cgggtaaagg cctggaatgg gttgctcgta  360tctacccgac caacggttac accaggtatg ccgattcagt taaaggtcgt ttcaccatct  420cggccgacac ttccaaaaac accgcttacc tccagatgaa ctccctgcgt gctgaagaca  480cagctgttta ttattgctcc cgttggggtg gtgacggttt ctacgctatg gactactggg  540gtcagggtac cctggtcacc gtctcctcag cctccaccaa gggcccatcg gtcttccccc  600tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc ctggtcaagg  660actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc  720acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc gtggtgactg  780tgccctccag cagcttgggc acccagacct acatctgcaa cgttaatcac aaacccagca  840acaccaaggt cgacaagaaa gttgagccca aatcttgcca tcaccaccat caccattaat  900aaccatggag aaaataaagt gaaacaaagc actattgcac tggcactctt accgttactg  960tttacccctg tgacaaaagc cgacatcgag ctcacccaat ccccgtcctc cctgtccgct 1020tccgttggcg accgtgttac catcacgtgt agggcctcgc aagacgtaaa caccgccgta 1080gcgtggtatc agcagaaacc cgggaaagct ccgaaactgc tgatctatag cgcttccttc 1140ctgtattccg gagttccgag caggttcagt ggttcccgtt ccggtaccga cttcaccctg 1200acgatatcct ccctccagcc ggaagacttc gctacctact actgtcaaca gcactacacc 1260accccgccga ccttcggtca gggtaccaaa ctcgagatca aacggactgt ggctgcacca 1320tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc ctctgttgtg 1380tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc 1440ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac 1500agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc 1560tgcgaagtca cccatcaggg cctgagttcg cccgtcacaa agagcttcaa ccgcggagag 1620tgctcttctg cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 1680cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 1740cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 1800cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 1860cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 1920cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 1980cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 2040cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 2100cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 2160cctgcaccag cccctgctcc tgctccagca cctgcaccag cacctgctcc agcaccagct 2220cctgcaccag cctgaagagc ttaagcttga cctgtgaagt gaaaaatggc gcacattgtg 2280cgacattttt tttgtctgcc gtttaccgct actgcgtcac ggatctccac gcgccctgta 2340gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 2400gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 2460ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 2520acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 2580agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 2640aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 2700cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 2760acaaaatatt aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc 2820ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 2880gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 2940cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 3000tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 3060tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 3120cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 3180tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 3240agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 3300ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 3360ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 3420aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 3480gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaattg atagactgga 3540tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 3600ttgctgataa atctggagcc ggtgagcgtg gctctcgcgg tatcattgca gcactggggc 3660cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 3720atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaggaat 3780taatgatgtc tcgtttagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg 3840aggtcggaat cgaaggttta acaacccgta aactcgccca gaagctaggt gtagagcagc 3900ctacattgta ttggcatgta aaaaataagc gggctttgct cgacgcctta gccattgaga 3960tgttagatag gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt 4020tacgtaataa cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag 4080tacatttagg tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct 4140ttttatgcca acaaggtttt tcactagaga atgcattata tgcactcagc gcagtggggc 4200attttacttt aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg 4260aaacacctac tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc 4320accaaggtgc agagccogcc ttcttattcg gccttgaatt gatcatatgc ggattagaaa 4380aacaacttaa atgtgaaagt gggtcttaaa agcagcataa cctttttccg tgatggtaac 4440ttcactagtt taaaaggatc taggtgaaga tcctttttga taatcccatg accaaaatcc 4500cttaacgtga gttttcgctc caccgagcgt cagaccccgt agaaaagacc aaaggatctt 4560cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4620cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4680tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 4740tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4800ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4860aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 4920cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 4980ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 5040agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 5100ctgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 5160acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg            5210SEQ ID NO: 59acccgacacc atcgaatggc cagatgatta attcctaatt tttgttgaca ctctatcatt   60gatagagtta ttttaccact ccctatcagt gatagagaaa agtgaaatga atagttcgac  120aaaaatctag ataacgaggg caaaaaatga aaaagacagc tatcgcgatt gcagtggcac  180tggctggttt cgctaccgta gcgcaggccg aagttaaact gcaggaatcc ggtggtggtc  240tggttcagcc aggtggttcc ctgcggctct cgtgtgctgc ttccggtttc aacatcaaag  300acacctacat ccactgggtt cgtcaggctc cgggtaaagg cctggaatgg gttgctcgta  360tctacccgac caacggttac accaggtatg ccgattcagt taaaggtcgt cccaccatct  420cggccgacac ttccaaaaac accgcttacc tccagatgaa ctccctgcgt gctgaagaca  480cagctgttta ttattgctcc cgttggggtg gtgacggttt ctocgctatg gactactggg  540gtcagggtac cctggtcacc gtctcctcag cctccaccaa gggcccatcg gtcttccccc  600tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc ctggtcaagg  660actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc  720acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc gtggtgactg  780tgccctccag cagcttgggc acccagacct acatctgcaa cgttaaccac aaacccagca  840acaccaaggt cgacaagaaa gttgagccca aatcttgcca tcaccaccat caccattaat  900aaccatggag aaaataaagt gaaacaaagc actattgcac tggcactctt accgttactg  960tttacccctg tgacaaaagc cgacatcgag ctcacccaat ccccgCcctc cctgtccgct 1020tccgttggcg accgtgtcac catcacgtgt agggcctcgc aagacgcaaa caccgccgta 1080gcgtggtatc agcagaaacc cgggaaagct ccgaaactgc tgatctatag cgcttccttc 1140ctgtattccg gagttccgag caggttcagt ggttcccgtt ccggtaccga cttcaccctg 1200acgatatcct ccctccagcc ggaagacttc gctacctact actgtcaaca gcactacacc 1260accccgccga ccttcggtca gggtaccaaa ctcgagatca aacggactgt ggctgcacca 1320tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc ctctgttgtg 1380tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc 1440ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac 1500agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc 1560tgcgaagtca cccatcaggg cctgagttcg cccgtcacaa agagcttcaa ccgcggagag 1620tgctcttctg ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 1680gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 1740gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 1800gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 1860gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 1920gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 1980gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 2040gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 2100gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 2160gcagctccag ccgctgcacc tgctgcagca cctgctgcag ctccagcagc tgctcctgca 2220gcagctccag cctgaagagc ttaagcttga cctgtgaagt gaaaaatggc gcacattgtg 2280cgacattttt tttgtctgcc gtttaccgct actgcgtcac ggatctccac gcgccctgta 2340gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 2400gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 2460ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 2520acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 2580agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 2640aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 2700cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 2760acaaaatatt aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc 2820ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 2880gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 2940cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 3000tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 3060tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 3120cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 3180tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 3240agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 3300ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 3360ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 3420aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 3480gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaattg atagactgga 3540tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 3600ttgctgataa atctggagcc ggtgagcgtg gctctcgcgg tatcattgca gcactggggc 3660cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 3720atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaggaat 3780taatgatgtc tcgtttagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg 3840aggtcggaat cgaaggttta acaacccgta aactcgccca gaagctaggt gtagagcagc 3900ctacattgta ttggcatgta aaaaataagc gggctttgct cgacgcctta gccattgaga 3960tgttagatag gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt 4020tacgtaataa cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag 4080tacatttagg tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct 4140ttttatgcca acaaggtttt tcactagaga atgcattata tgcactcagc gcagtggggc 4200attttacttt aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg 4260aaacacctac tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc 4320accaaggtgc agagccagcc ttcttattcg gccttgaatt gatcatatgc ggattagaaa 4380aacaacttaa atgtgaaagt gggtcttaaa agcagcataa cctttttccg tgatggtaac 4440ttcactagtt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4500cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4560cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4620cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4680tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 4740tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4800ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4860aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 4920cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 4980ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 5040agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 5100ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 5160acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg            5210

Example 18: Measurement of the Hydrodynamic Volume for the RecombinantFusion Protein Between a Fab Fragment and a Genetically Encoded P1A1 orP1A3 Polypeptide/Polymer by Analytical Gel Filtration

SEC was carried out on a Superdex S200 HR 10/300 GL column (GEHealthcare Europe, Freiburg, Germany) at a flow rate of 1 ml/min usingan Äkta Purifier 10 system (GE Healthcare) with PBS as running buffer.250 μl samples of the Fab-P1A1(200) and Fab-P1A3(200) fusion proteins,which were similarly produced and purified (FIG. 9) as described forFab-PA#1(200) in Example 4, were individually applied at a concentrationof 0.25 mg/ml in PBS. Both proteins eluted in a single homogenous peakas shown in FIG. 10.

As result, the fusion proteins with the 200 residue P1A1 or P1A3polymers/polypeptides exhibited significantly larger sizes than thecorresponding unfused Fab fragment. The apparent size increase forFab-P1A1(200) and Fab-P1A3(200) was 5.8-fold and 5.2-fold, respectively,compared with the Fab fragment (cf. FIG. 4B) whereas the true mass wasonly larger by 1.4-fold and 1.3-fold. This observation clearly indicatesa much increased hydrodynamic volume conferred to the biologicallyactive Fab fragment by the biosynthetic P1A1 and P1A3 polypeptidesegments according to this invention.

Example 19: Detection of Random Coil Conformation for the BiosyntheticP1A1 and P1A3 Polymers/Polypeptides Fused to a Fab Fragment Via CircularDichroism (CD) Spectroscopy

CD spectra for Fab-P1A1(200) and Fab-P1A3(200) were recorded asdescribed in Example 8 using 4.2 and 6.5 μM protein solutions,respectively, prepared similary as described in Example 4 using 50 mMK₂SO₄, 20 mM K-phosphate pH 7.5 as buffer.

The spectra for the Fab-P1A1(200) and Fab-P1A3(200) fusion proteinsrevealed a significant fraction of random coil conformation (FIG. 11A).To analyze the spectroscopic contribution by the Pro/Ala polypeptidesegment in greater detail the molar difference CD spectrum with respectto the unfused Fab fragment (see Example 8) was calculated (FIG. 11B) bysubtracting the latter spectrum from the one for Fab-P1A1(200) andFab-P1A3(200), respectively, after normalization to the same molarconcentration. As result, a strong minimum at a wavelength ofapproximately 200 nm, which is characteristic of random coilconformation, was observed. Thus, the P1A1 and the P1A3 sequences aspart of the recombinant fusion protein appear to be present in randomcoil conformation under physiological buffer conditions.

Example 20: Construction of PSUMO-PA#1(200) as Expression Vector for aHis(6)-SUMO-PA#1(200) Fusion Protein

For the construction of an expression plasmid encoding a six-residueHis-tag and the small ubiquitin-like modifier (SUMO) protein (Panavas(2009) Methods Mol. Biol. 497: 303-17) fused to a 200 residue PA#1sequence repeat, the SUMO protein) from Saccharomyces cerevisiae [alsoknown as Smt3p; Uniprot: Q12306] was amplified via polymerase chainreaction (PCR) from a cloned cDNA. The 5′-primer introduced an NdeIrestriction site, containing a Met start codon (ATG) and an additionalLys codon, as well as the His6-tag encoding sequence while the 3′-primerintroduced a HindIII and SapI restriction site into the PCR product. Theresulting DNA fragment was digested with NdeI and HindIII and ligatedwith a correspondingly digested derivative of the plasmid pSA1 (Schmidt(1994) J. Chromatogr. 676: 337-345), wherein the SapI restriction sitehad been eliminated by silent mutation. The resulting plasmid was cutwith SapI, dephosphorylated with shrimp alkaline phosphatase, andligated with the gene fragment encoding the 200 residue PA#1 polypeptidesegment excised from the plasmid pFab-PA#1(200) (described in Example 2)by restriction digest with SapI (in an analogous way as exemplified inFIG. 2E). The resulting plasmid was designated pSUMO-PA#1(200) (SEQ IDNO: 60) and is depicted in FIG. 12A.

Example 21: Bacterial Expression and Isolation of a Genetically EncodedPA#1(200) Polymer/Polypeptide

The PA#1(200) polypeptide (calculated mass: 16.1 kDa) was initiallyproduced as fusion protein with the small ubiquitin-like modifier (SUMO)protein (calculated mass: 12.2 kDa) in the cytoplasm of E. coli BLR(DE3)(NEB, Ipswich, Mass., USA) harboring the expression plasmidpSUMO-PA#1(200) (described in Example 21) together with the plasmidpLysE (Studier (1991) J. Mol. Biol. 219: 37-44), which suppresses thethe T7 promoter. Bacterial production was performed at 30° C. in shakeflask cultures with 2 L LB medium containing 2.5 g/L D-glucose, 0.5 g/LL-proline, 100 mg/l ampicillin, and 30 mg/l chloramphenicol. Recombinantgene expression was induced by addition ofisopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of0.5 mM. Bacteria were harvested 3 h after induction, resuspended in 100mM NaCl, 40 mM Na-phosphate pH 7.5 and lysed using a French pressurecell (Thermo Scientific, Waltham, Mass., USA). After centrifugation (15min, 15000 g) of the lysate no inclusion bodies were observed.

The supernatant containing the soluble fusion protein was incubated at70° C. for 15 min and centrifuged (15 min, 15000 g) to remove thermallyunstable host cell proteins. The His(6)-SUMO-PA#1(200) fusion proteinwas purified from the supernatant via IMAC (Skerra (1994) Gene 141:79-84) using a 12 ml Ni₂ ⁺ charged HisTrap high performance column (GEHealthcare) connected to an Äkta purifier system (GE Healthcare) andeluted with an imidazole gradient from 0 to 150 mM in 500 mM NaCl, 40 mMNa-phosphate pH 7.5. After a subsequent preparative SEC step ahomogeneous preparation of the His(6)-SUMO-PA#1(200) fusion protein(FIG. 12B) with a yield of approximately 5 mg per 1 L bacterial culturewith OD550=1 was obtained. Protein concentration was determinedaccording to the absorption at 280 nm using a calculated extinctioncoefficient (Gill (1989) loc. cit) of 1280 M⁻¹ cm⁻¹ for theHis(6)-SUMO-PA#1(200) polypeptide fusion. Note that the PA#1(200)polypeptide segment does not contribute to the absorption at 280 nm dueto its lack of aromatic or sulfur-containing amino acid side chains.

The biosynthetic PA#1(200) polypeptide was liberated from the fusionprotein by site specific proteolytic cleavage (downstream of a Gly-Glymotif preceding the Pro/Ala polypeptide segment) with 2 U/mgUb1-specific protease 1 from Saccharomyces cerevisiae (Invitrogen,Carlsbad, Calif., USA) for 1 h at 30° C. in cleavage buffer (0.2 w/v %Igepal, 1 mM DTT, 150 mM NaCl, 50 mM Tris-HCl pH 8.0). The cleavageprocess was checked by SDS-PAGE (FIG. 12B) using a high molarity Trisbuffer system (Fling (1986) Anal. Biochem. 155: 83-88). In order toremove the cleaved His(6)-SUMO protein, residual uncleaved fusionprotein, and also the SUMO protease, all carrying the His₆-tag, thereaction mixture was subjected to another IMAC using a 5 ml Ni₂ ⁺charged HisTrap high performance column (GE Healthcare) and 500 M NaCl,20 mM phosphate, pH 7.5 as running buffer. This time the flow-throughcontained the pure biosynthetic PA#1(200) polypeptide (FIG. 13E). Notethat the biosynthetic PA#1(200) polypeptide/polymer (SEQ ID NO: 61)prepared in this manner comprises altogether 201 amino acid residues,which arise from the encoded combined gene product of 10 ligateddouble-stranded oligodeoxynucleotide building blocks, each encoding 20amino acid residues, as shown in FIG. 1, and an additional Ala residueencoded by the triplet DNA overhang of the downstream SapI restrictionsite that was used for cloning.

Example 22: Preparation and Characterization of Small Molecule/DrugConjugates with PA#1(200)

The unpurified proteolytic cleavage reaction mixture of theHis(6)-SUMO-PA#1(200) fusion protein from Example 21 was twice dialysedat 4° C. against 50 mM NaHCO₃ pH 8.3 and incubated at room temperaturefor 1 h after mixing with a 10-fold molar excess of a solution of6-[fluorescein-5(6)-carboxamido] hexanoic acid N-hydroxysuccinimideester (Fluorescein-NHS ester; Sigma-Aldrich) in dry dimethylformamide(DMF). To this end, 200 μl of a 2.5 mg/ml solution of theHis(6)-SUMO-PA#1(200) cleavage mixture was added to 17.6 μl of a 10 mMsolution of Fluorescein-NHS ester dissolved in DMF. The resultingmixture was incubated at room temperature for 1 h and applied to IMAC asdescribed in Example 21 to remove the cleaved His(6)-SUMO protein,residual uncleaved fusion protein, and the SUMO protease and furtherpurified by preparative SEC on a Superdex S200 10/300 GL columnequilibrated with PBS at a flow rate of 0.5 ml/min.

Samples from the different steps were then analysed via analytical SECon a Superdex S200 10/300 GL column equilibrated with PBS at a flow rateof 0.5 ml/min. The SUMO protein was detected via its aromatic sidechains at 280 nm and the peptide bonds, including those of the Pro/Alapolypeptide or polypeptide segment, were detected at 225 nm whilefluorescein was detected at 494 nm (FIG. 13 A-G). For comparison, UV/VISspectra of a solution of free fluorescein (Sigma-Aldrich) and offractions from each distinct peak detected in the SEC were measuredusing a Lambda 9 instrument (Perkin Elmer, Waltham, Mass., USA) (FIG. 13H-K). For size calibration of the chromatography column (FIG. 13L), 250μl of an appropriate mixture of the following globular proteins(Sigma-Aldrich) were applied in PBS at concentrations between 0.2 and0.5 mg/ml: aprotinin, 6.5 kDa; cytochrome c, 12.4 kDa; carbonicanhydrase, 29.0 kDa; bovine serum albumin, 66.3 kDa; alcoholdehydrogenase, 150 kDa; β-amylase, 200 kDa; apo-ferritin, 440 kDa.

As result, after coupling of the biosynthetic PA#1(200)polypeptide/polymer with Fluorescein-NHS ester a macromolecularconjugate was isolated via IMAC and SEC that essentially exhibits thesize properties of the PA#1(200) polypeptide/polymer and thespectroscopic signature of the small molecule, i.e. the fluoresceingroup. This demonstrates that the small molecule was successfullycoupled to the biosynthetic Pro/Ala polypeptide/polymer, which accordingto this invention dramatically increases the hydrodynamic volume of theconjugated small molecule drug or compound.

To prepare a similar conjugate between the biosynthetic Pro/Alapolypeptide/polymer and the plant steroid digoxigenin, 0.1 mg of thepurified PA#1(200) polypeptide from Example 21 was dialysed against 50mM NaHCO₃ pH 8.3 as described above. The concentration of purifiedPA#1(200) polypeptide was determined according to the absorption at 205nm (Gill (1989) loc. cit). The PA#1(200) polypeptide was coupled with a10-fold molar excess of digoxigenin-3-O-methylcarbonyl-ε-aminocaproicacid NHS ester (DIG-NHS ester; Roche Diagnostics, Mannheim, Germany).For this purpose, 100 μl of a 1 mg/ml solution of the purified PA#1(200)polypeptide in 50 mM NaHCO₃ pH 8.3 was added to 2 μl of a 30 mM solutionof DIG-NHS ester dissolved in dry DMF and the reaction mix was incubatedfor 1 h at room temperature. The resulting solution of the conjugate waspurified using a Zeba™ spin desalting column with a cutoff of 7 kDa(Thermo Scientific), twice dialysed against 10 mM ammonium acetatebuffer pH 6.8 and analysed via ESI mass spectrometry on a Q-Tof Ultimainstrument (Waters, Eschbronn, Germany) using the positive ion mode. Asresult, the spectrum of the Digoxigenin-PA#1(200) conjugate revealed amass of 16671.4 Da, which essentially coincides with the calculated massof 16670.6 Da (FIG. 13M). This clearly demonstrates that a biosyntheticPro/Ala polypeptide/polymer, in particular PA#1(200), can be efficientlyconjugated with a small molecule drug.

The present invention relates to and refers to the following exemplifiedsequences, whereby the appended sequence listing is presented as part ofthe description and is, accordingly a part of this specification.

SEQ ID NO: 1 shows the amino acid sequence of PA#1.

SEQ ID NO: 2 shows the amino acid sequence of PA#2.

SEQ ID NO: 3 shows the amino acid sequence of PA#3.

SEQ ID NO: 4 shows the amino acid sequence of PA#4.

SEQ ID NO: 5 shows the amino acid sequence of PA#5.

SEQ ID NO: 6 shows the amino acid sequence of PA#6.

SEQ ID NO: 7 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 8 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 9 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 10 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 11 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 12 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 13 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 14 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 15 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 16 shows an amino acid sequence of a circular permutatedversion of SEQ ID NO: 1.

SEQ ID NO: 17 shows a nucleic acid sequence of the upper/coding strandoligodeoxynucleotide used for the generation of building block PA#1.

SEQ ID NO: 18 shows a nucleic acid sequence of lower/non-coding strandoligodeoxynucleotide used for the generation of the building block forPA#1.

SEQ ID NO: 19 shows a nucleic acid sequence stretch (upper/codingstrand) around the C-terminus of the immunoglobulin light chain of anantibody Fab fragment as encoded on pASK88-Fab-2xSapI.

SEQ ID NO: 20 shows a nucleic acid sequence stretch (lower/non-codingstrand) around the C-terminus of the immunoglobulin light chain of anantibody Fab fragment as encoded on pASK88-Fab-2xSapI.

SEQ ID NO: 21 shows an amino acid sequence of the C-terminus of thelight chain of the Fab fragment as encoded on pASK88-Fab-2xSapI.

SEQ ID NO: 22 shows the nucleic acid sequence of pASK88-Fab-2xSapI.

SEQ ID NO: 23 shows a nucleic acid sequence stretch (upper/codingstrand) encoding amino acid sequence of the C-terminus of the Fab lightchain after insertion of one PA#1(20) polymer.

SEQ ID NO: 24 shows a nucleic acid sequence (lower/non-coding strand)for an amino acid stretch of the C-terminus of an Fab light chain afterinsertion of one PA#1(20) polymer.

SEQ ID NO: 25 shows an amino acid sequence stretch of the C-terminus ofan Fab light chain after insertion of one PA#1(20) polymer.

SEQ ID NO: 26 shows the amino acid sequence of the Fab heavy chain asencoded on pFab-PA#1(200).

SEQ ID NO: 27 shows the amino acid sequence of the Fab light chain fusedwith the PA#1(200) polymer as encoded on pFab-PA#1(200).

SEQ ID NO: 28 shows the nucleic acid sequence of pFab-PA#1(200).

SEQ ID NO: 29 shows the nucleic acid sequence (upper/coding strand)encoding the amino acid sequence of the N-terminus of INFa2b andStrep-tag II (only the last two amino acids).

SEQ ID NO: 30 shows a nucleic acid sequence (lower/non-coding strand)encoding amino acid sequence of the N-terminus of INFa2b and Strep-tagII (only the last two amino acids).

SEQ ID NO: 31 shows the amino acid sequence of the C-terminus ofStrep-tag II and the N-terminus of INFa2b.

SEQ ID NO: 32 shows the nucleic acid sequence of pASK-IFNa2b.

SEQ ID NO: 33 shows a nucleic acid sequence stretch (upper/codingstrand) encoding the C-terminus of Strep-tag II and the N-terminus ofIFNa2b after insertion of one PA#1 polymer sequence cassette.

SEQ ID NO: 34 shows a nucleic acid sequence stretch (lower/non-codingstrand) of the C-terminus of Strep-tag II and the N-terminus of IFNa2bafter insertion of one PA#1 polymer sequence cassette.

SEQ ID NO: 35 shows an amino acid sequence stretch of the C-terminus ofStrep-tag II and the N-terminus of IFNa2b ater fusion with one PA#1polymer cassette.

SEQ ID NO: 36 shows the amino acid sequence of IFNa2b and Strep-tag IIfused with the PA#1(200) polymer as encoded on pPA#1(200)-IFNa2b.

SEQ ID NO: 37 shows the nucleic acid sequence of pPA#1(200)-IFNa2b.

SEQ ID NO: 38 shows a nucleic acid sequence stretch (upper/codingstrand) on pASK75-His6-hGH encoding the amino acid sequence around theN-terminus of His6-hGH.

SEQ ID NO: 39 shows a nucleic acid sequence stretch (lower/non-codingstrand) on pASK75-His6-hGH encoding the amino acid sequence around theN-terminus of hGH.

SEQ ID NO: 40 shows an amino acid sequence stretch of the N-terminus ofHis6-hGH as encoded on pASK75-His6-hGH.

SEQ ID NO: 41 shows the nucleic acid sequence of pASK75-His6-hGH.

SEQ ID NO: 42 shows a nucleic acid sequence (upper/coding-strand)stretch encoding amino acid sequence of the N-terminus of His6-hGH afterinsertion of the PA#1(20) polymer.

SEQ ID NO: 43 shows a nucleic acid sequence (lower/non-coding strand)encoding the N-terminus of hGH after insertion of one PA#1 polymersequence cassette.

SEQ ID NO: 44 shows the amino acid sequence of the N-terminus ofHis6-hGH after insertion of the PA#1(20) polymer.

SEQ ID NO: 45 shows the amino acid sequence of mature His6-PA#1(200)-hGHas encoded on pASK75-His6-PA#1(200)-hGH.

SEQ ID NO: 46 shows the nucleic acid sequence ofpASK75-His6-PA#1(200)-hGH.

SEQ ID NO: 47 shows the amino acid sequence of His6-PA#1(200)-hGH asencoded on pCHO-PA#1(200)-hGH.

SEQ ID NO: 48 shows the nucleic acid sequence of pCHO-PA#1(200)-hGH.

SEQ ID NO: 49 shows the nucleic acid sequence of pCHO-hGH.

SEQ ID NO: 50 shows the nucleic acid sequence of pCHO.

SEQ ID NO: 51 shows the amino acid sequence of P1A1.

SEQ ID NO: 52 shows the nucleic acid sequence of upper/coding strandoligodesoxynucleotide used for the generation of the building block forP1A1.

SEQ ID NO: 53 shows the nucleic acid sequence of lower/non-coding strandoligodesoxynucleotide used for the generation of the building block forP1A1.

SEQ ID NO: 54 shows the nucleic acid sequence of upper/coding strandoligodesoxynucleotide used for the generation of the building block forP1A3.

SEQ ID NO: 55 shows the nucleic acid sequence of lower/non-coding strandoligodesoxynucleotide used for the generation of the building block forP1A3.

SEQ ID NO: 56 shows the amino acid sequence of the Fab light chain fusedwith the P1A1(200) polymer as encoded on pFab-P1A1(200).

SEQ ID NO: 57 shows the amino acid sequence of the Fab light chain fusedwith the P1A3(200) polymer as encoded on pFab-P1A3(200).

SEQ ID NO: 58 shows the nucleic acid sequence of pFab-P1A1(200).

SEQ ID NO: 59 shows the acid sequence of pFab-P1A3(200).

SEQ ID NO: 60 shows the nucleic acid sequence of pSUMO-PA#1(200).

SEQ ID NO: 61 shows the PA#1(200) polypeptide/polymer used for thepreparation of drug conjugates (made by ligation of 10 20mer encodinggene cassettes, including one additional C-terminal Ala residueresulting from the downstream ligation site.

The invention claimed is:
 1. A drug conjugate comprising (i) abiosynthetic random coil polypeptide or polypeptide segment comprisingan amino acid sequence consisting of proline and alanine amino acidresidues, wherein said amino acid sequence consists of at least 150proline (Pro) and alanine (Ala) amino acid residues, and (ii) a drugselected from the group consisting of (a) a biologically active proteinor a polypeptide that comprises or that is an amino acid sequence thathas or mediates a biological activity and (b) a small molecule drug. 2.The drug conjugate according to claim 1, wherein said random coilpolypeptide or polypeptide segment comprises an amino acid sequenceconsisting of 150 to 3000 amino acid residues.
 3. The drug conjugateaccording to claim 1, wherein said proline residues constitute more than10% and less than 75% of the amino acid sequence.
 4. The drug conjugateaccording to claim 1, wherein said random coil polypeptide orpolypeptide segment comprises a plurality of amino acid repeats, whereinsaid repeat consists of proline and alanine residues and wherein no morethan 6 consecutive amino acid residues are identical.
 5. The drugconjugate according to claim 1, wherein said random coil polypeptide orpolypeptide segment comprises an amino acid sequence selected from thegroup consisting of (SEQ ID NO: 1) AAPAAPAPAAPAAPAPAAPA; (SEQ ID NO: 2)AAPAAAPAPAAPAAPAPAAP; (SEQ ID NO: 3) AAAPAAAPAAAPAAAPAAAP;(SEQ ID NO: 4) AAPAAPAAPAAPAAPAAPAAPAAP; (SEQ ID NO: 5)APAAAPAPAAAPAPAAAPAPAAAP; (SEQ ID NO: 6) AAAPAAPAAPPAAAAPAAPAAPPA; and(SEQ ID NO: 51) APAPAPAPAPAPAPAPAPAP

or circular permuted versions or (a) multimers(s) of these sequences asa whole or parts of these sequences.
 6. The drug conjugate according toclaim 1, wherein said polypeptide with biological activity, saidbiologically active protein or said polypeptide that comprises or thatis an amino acid sequence that has or that mediates a biologicalactivity is selected from the group consisting of binding proteins,antibody fragments, cytokines, growth factors, hormones or enzymes. 7.The drug conjugate of claim 6, wherein said polypeptide with biologicalactivity is a binding protein and wherein said binding molecule isselected from the group consisting of antibodies, Fab fragments, F(ab′)₂fragments, CDR-derived peptidomimetics, single chain variable fragments(scFv), domain antibodies, lectins, immunoglobulin domains, fibronectindomains, protein A domains, SH3 domains, ankyrin repeat domains, andlipocalins.
 8. The drug conjugate of claim 1, wherein said biologicallyactive protein is selected from the group consisting of granulocytecolony stimulating factor, human growth hormone, alpha-interferon,beta-interferon, gamma-interferon, tumor necrosis factor,erythropoietin, coagulation factor VIII, gp120/gp160, soluble tumornecrosis factor I and II receptor, reteplase, exendin-4, anakinra,interleukin-2, neutrophil gelatinase-associated lipocalin,follicle-stimulating hormone, glucocerebrosidase, thymosin alpha 1,glucagon, somatostatin, adenosine deaminase, interleukine 11,coagulation factor VIIa, coagulation factor IX, hematide,lambda-interferone, leptin, interleukine-22 receptor subunit alpha(IL-22ra), interleukine-22, hyaluronidase, fibroblast growth factor 18,fibroblast growth factor 21, glucagon-like peptide 1, osteoprotegerin,IL-18 binding protein, growth hormone releasing factor, soluble TACIreceptor, thrombospondin-1, soluble VEGF receptor Flt-1, and IL-4mutein.
 9. The drug conjugate according to claim 1, whereby saidbiosynthetic random coil polypeptide or polypeptide mediates anincreased in vivo and/or in vitro stability of said drug conjugate. 10.The drug conjugate according to claim 9, wherein said increased in vivostability is a prolonged plasma half-life of said drug conjugate whencompared to the stability of a control polypeptide or a controlconjugate lacking said random coil random coil polypeptide orpolypeptide segment.
 11. The drug conjugate according to claim 1,wherein said small molecule is selected from the group consisting ofdigoxigenin, fluorescein, doxorubicin, calicheamicin, camptothecin,fumagillin, dexamethasone, geldanamycin, paclitaxel, docetaxel,irinotecan, cyclosporine, buprenorphine, naltrexone, naloxone,vindesine, vancomycin, risperidone, aripiprazole, palonosetron,granisetron, cytarabine NX1838, leuprolide, goserelin, buserelin,octreotide, teduglutide, cilengitide, abarelix, enfuvirtide, ghrelin,alpha 4 integrin inhibitors, antisense nucleic acids, small interferenceRNAs, micro RNAs, steroids, DNA or RNA aptamers and peptides and/orpeptidomimetics.
 12. A composition comprising the drug conjugateaccording to claim
 1. 13. The composition according to claim 12 which isa pharmaceutical composition or a diagnostic composition, optionallyfurther comprising a pharmaceutically acceptable carrier(s).
 14. Thedrug conjugate according to claim 1, wherein the biologically activeprotein is selected from the group consisting of binding proteins,antibody fragments, cytokines, growth factors, hormones and enzymes.